Method for controlling the Young's modulus of a three-dimensional tissue, method for manufacturing a three-dimensional tissue, and three-dimensional tissue

Abstract

To provide a method for controlling the Young's modulus of a three-dimensional tissue, a method for manufacturing a three-dimensional tissue, and a three-dimensional tissue, in which a three-dimensional tissue is manufactured using a highly biocompatible scaffolding material. [Solution] A method for controlling the Young's modulus of a three-dimensional tissue including cells and extracellular matrix by adjusting the average diameter of the extracellular matrix.

Description

Technical Field

[0001] The present invention relates to a method for controlling the Young's modulus of a three-dimensional tissue, a method for manufacturing a three-dimensional tissue, and a three-dimensional tissue.

Background Art

[0002] In recent years, techniques for constructing three-dimensional tissues of cells in vitro have been developed. When producing a tissue mimicking a living tissue, it is preferable to use a scaffold material with high biocompatibility. The present inventors have hitherto proposed a method for manufacturing a three-dimensional tissue including arranging cells coated with a film containing collagen three-dimensionally to form a three-dimensional tissue (Patent Document 1), a method for manufacturing a three-dimensional tissue including forming coated cells having a film formed on the surface of cells, and arranging the coated cells three-dimensionally, wherein the formation of the coated cells includes immersing cells in a liquid containing a film component and separating the immersed cells and the liquid containing the film component with a liquid-permeable membrane (Patent Document 2), etc. Further, the present inventors have proposed a method for producing a three-dimensional tissue having a higher collagen concentration than conventional ones using homogenized collagen (Patent Document 3).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] Incidentally, it is known that the "hardness" of tissues in actual living organisms varies depending on the location within the body or the type of lesion. For example, cancerous tissue tends to be harder than normal tissue. Since the hardness of the tissue can be one of the factors that create the characteristics of organs and disease states, it is important to reproduce the original hardness when creating tissue models that mimic living tissue.

[0005] However, to date, there have been no reports on methods for adjusting the stiffness of tissue bodies manufactured using highly biocompatible scaffold materials to a desired stiffness, in accordance with the tissue to be reproduced. For example, a method is known to control the stiffness of the cell growth environment by culturing cells in polyacrylamide gel (RWTilghman et al., PLoS One, 5(9) e12905 (2010)). However, since polyacrylamide gel is not a substance that exists in living organisms, it is undesirable from the standpoint of mimicking biological tissue.

[0006] Therefore, one aspect of the present invention aims to provide a method for controlling the Young's modulus of a three-dimensional tissue in a three-dimensional tissue manufactured using a highly biocompatible scaffolding material. Another aspect of the present invention aims to provide a method for manufacturing a three-dimensional tissue having a desired Young's modulus. Yet another aspect of the present invention aims to provide a three-dimensional tissue with a Young's modulus of 20 kPa or higher. [Means for solving the problem]

[0007] The inventors found that when comparing compositions containing an extracellular matrix with a small average diameter and an extracellular matrix with a large average diameter at the same concentration in an aqueous medium, the composition containing the extracellular matrix with a small average diameter exhibited a higher Young's modulus.

[0008] Therefore, the present invention relates to, for example, the following inventions. [1] A method for controlling the Young's modulus of a three-dimensional tissue including cells and extracellular matrix by adjusting the average diameter of the extracellular matrix. [2] The method according to [1], wherein the extracellular matrix is ​​a polypeptide having an RGD sequence. [3] The method according to [2], wherein the extracellular matrix is ​​a biocompatible material in which the molecules are identical to those present in the living body. [4] The method according to any one of [1] to [3], wherein the extracellular matrix includes an exogenous fragmented extracellular matrix. [5] The method according to any one of [1] to [4], wherein the extracellular matrix contains collagen. [6] The method according to any one of [1] to [5], comprising adjusting the average diameter of the extracellular matrix to 20 nm or more and 1000 nm or less. [7] The method according to any one of [1] to [6], comprising preparing a three-dimensional tissue to contain fragmented extracellular matrix of different average diameters. [8] The method according to [7], comprising preparing an extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less, and an extracellular matrix having an average diameter of 1.5 μm or more and 8.5 μm or less. [9] The method according to [8], wherein an extracellular matrix with an average diameter of 20 nm to 1000 nm and an extracellular matrix with an average diameter of 1.5 μm to 8.5 μm are used in a weight ratio of 1:100 to 100:1.

[10] The method according to [9], wherein an extracellular matrix with an average diameter of 20 nm to 1000 nm is used in greater weight than an extracellular matrix with an average diameter of 1.5 μm to 8.5 μm.

[11] The method according to any one of [1] to

[10] , wherein the Young's modulus of the three-dimensional structure is 20 kPa to 250 kPa.

[12] A step of preparing an extracellular matrix whose average diameter is adjusted to match the desired Young's modulus of the three-dimensional tissue, A step of mixing the above extracellular matrix with cells in an aqueous medium to obtain a cell suspension. The process includes the step of incubating the above cell suspension. A method for manufacturing a three-dimensional tissue.

[13] The method according to

[12] , wherein the extracellular matrix is ​​a polypeptide having an RGD sequence.

[14] The method according to

[13] , wherein the extracellular matrix is ​​a biocompatible material in which the extracellular matrix is ​​the same molecule as a molecule present in the living body.

[15] The method according to any one of

[12] to

[14] , wherein the extracellular matrix includes an exogenous fragmented extracellular matrix.

[16] The method according to any one of

[12] to

[15] , wherein the extracellular matrix contains collagen.

[17] The method according to any one of

[12] to

[16] , wherein the extracellular matrix with the average diameter adjusted above includes an extracellular matrix with an average diameter of 20 nm or more and 1000 nm or less.

[18] The method according to any one of

[12] to

[17] , wherein the step of preparing the fragmented extracellular matrix comprises adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters.

[19] The method according to

[18] , comprising preparing a three-dimensional tissue to include fragmented extracellular matrix having an average diameter of 20 nm to 1000 nm and fragmented extracellular matrix having an average diameter of 1.5 μm to 8.5 μm.

[20] The method according to any one of

[12] to

[19] , further comprising the step of applying an external force to the cell suspension to accumulate the cells and the extracellular matrix between the step of obtaining the cell suspension and the step of incubating the cell suspension.

[21] The method according to any one of

[12] to

[20] , wherein the Young's modulus of the three-dimensional structure is 20 kPa to 250 kPa.

[22] A cell comprising an extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less, The fragmented extracellular matrix is ​​arranged between the above cells. The above extracellular matrix is ​​a biocompatible material whose molecules are identical to those present in living organisms. A three-dimensional tissue with a Young's modulus of 20 kPa or higher. 〔23〕 The three-dimensional tissue body according to 〔22〕, wherein the extracellular matrix is a polypeptide having an RGD sequence. 〔24〕 The three-dimensional tissue body according to 〔22〕 or 〔23〕, wherein the extracellular matrix contains an exogenous fragmented extracellular matrix. 〔25〕 The three-dimensional tissue body according to any one of 〔19〕 to 〔24〕, wherein the content of the extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less is 5% by weight or more and 90% by weight or less based on the total weight of the three-dimensional tissue body. 〔26〕 The three-dimensional tissue body according to any one of 〔19〕 to 〔25〕, further comprising an extracellular matrix having an average diameter of 1.5 μm or more and 8.5 μm or less. 〔27〕 A three-dimensional tissue body forming agent comprising an extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less and an aqueous medium, wherein the Young's modulus of the three-dimensional tissue body is 20 kPa or more.

[0009] Furthermore, the present invention relates to, for example, the following inventions. 〔1〕 A method for controlling the Young's modulus of a three-dimensional tissue body containing cells and an extracellular matrix by adjusting the average diameter of the fragmented extracellular matrix in the production of the three-dimensional tissue body. 〔2〕 The method according to 〔1〕, further comprising adjusting the content of the fragmented extracellular matrix. 〔3〕 The method according to 〔1〕 or 〔2〕, wherein the fragmented extracellular matrix is a polypeptide having an RGD sequence. 〔4〕 The method according to 〔3〕, wherein the fragmented extracellular matrix is a biocompatible material that is the same molecule as a molecule present in vivo. 〔5〕 The method according to any one of 〔1〕 to 〔4〕, wherein the fragmented extracellular matrix contains collagen. 〔6〕 The method according to any one of 〔1〕 to 〔5〕, comprising adjusting the average diameter of the fragmented extracellular matrix to 20 nm or more and 1000 nm or less. 〔7〕 The method according to any one of 〔1〕 to 〔5〕, which includes adjusting the average diameter of the fragmented extracellular matrix so that the three-dimensional tissue body includes fragmented extracellular matrices with different average diameters. 〔8〕 The method according to 〔7〕, which further includes adjusting the content of the fragmented extracellular matrices with different average diameters in the three-dimensional tissue body. 〔9〕 Adjusting so that the three-dimensional tissue body includes fragmented extracellular matrices with different average diameters and adjusting the content of the fragmented extracellular matrices with different average diameters in the three-dimensional tissue body are increasing the Young's modulus of the three-dimensional tissue body by increasing the content of the fragmented extracellular matrix with a small average diameter in the three-dimensional tissue body, or The method according to 〔8〕, which includes decreasing the Young's modulus of the three-dimensional tissue body by decreasing the content of the fragmented extracellular matrix with a small average diameter in the three-dimensional tissue body. 〔10〕 The method according to any one of 〔7〕 to 〔9〕, wherein the fragmented extracellular matrices with different average diameters include a fragmented extracellular matrix with an average diameter of 1000 nm or less and a fragmented extracellular matrix with an average diameter of more than 1000 nm. 〔11〕 The method according to any one of 〔7〕 to 〔9〕, wherein the fragmented extracellular matrices with different average diameters include a fragmented extracellular matrix with an average diameter of 20 nm or more and 1000 nm or less and a fragmented extracellular matrix with an average diameter of 1.5 μm or more and 8.5 μm or less. 〔12〕 The method according to any one of 〔8〕 to 〔11〕, wherein the fragmented extracellular matrix with a smaller average diameter and the fragmented extracellular matrix with a larger average diameter are used in a ratio of 1:100 to 100:1 in terms of weight conversion. 〔13〕 The method according to any one of 〔1〕 to 〔12〕, wherein the Young's modulus of the three-dimensional tissue body is 20 kPa to 250 kPa. 〔14〕 A step of preparing a fragmented extracellular matrix with an adjusted average diameter according to the desired Young's modulus of the three-dimensional tissue body, A step of mixing the extracellular matrix with cells in an aqueous medium to obtain a cell suspension, The process includes the step of incubating the above cell suspension. A method for manufacturing a three-dimensional tissue.

[15] The method according to

[14] , wherein the step of preparing the fragmented extracellular matrix is ​​the step of preparing a fragmented extracellular matrix whose average diameter and content are adjusted to match the desired Young's modulus of the three-dimensional tissue.

[16] The method according to

[14] or

[15] , wherein the fragmented extracellular matrix is ​​a polypeptide having an RGD sequence.

[17] The method according to

[16] , wherein the fragmented extracellular matrix is ​​a biocompatible material in which the molecules are identical to those present in the body.

[18] The method according to any one of

[14] to

[17] , wherein the fragmented extracellular matrix contains collagen.

[19] The method according to any one of

[14] to

[18] , wherein the fragmented extracellular matrix with adjusted average diameter includes a fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm.

[20] The method according to any one of

[14] to

[19] , wherein the step of preparing the fragmented extracellular matrix comprises adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters.

[21] The method according to

[20] , further comprising adjusting the content of the fragmented extracellular matrix of different average diameters in the three-dimensional tissue.

[22] Adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters and adjusting the content of the fragmented extracellular matrix of different average diameters in the three-dimensional tissue, By increasing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue, the Young's modulus of the three-dimensional tissue can be increased, or The method according to

[21] , comprising reducing the Young's modulus of a three-dimensional tissue by reducing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue.

[23] The method according to any one of

[20] to

[22] , wherein the fragmented extracellular matrix of different average diameters includes a fragmented extracellular matrix with an average diameter of 1000 nm or less and a fragmented extracellular matrix with an average diameter of more than 1000 nm.

[24] The method according to any one of

[20] to

[23] , wherein the fragmented extracellular matrix of different average diameters includes a fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm and a fragmented extracellular matrix with an average diameter of 1.5 μm to 8.5 μm.

[25] The method according to any of

[21] to

[24] , wherein the fragmented extracellular matrix with a smaller average diameter and the fragmented extracellular matrix with a larger average diameter are used in a ratio of 1:100 to 100:1 by weight.

[26] The method according to any one of

[14] to

[25] , wherein the Young's modulus of the three-dimensional structure is 20 kPa to 250 kPa.

[27] A cell comprising a fragmented extracellular matrix having an average diameter of 20 nm to 1000 nm, The fragmented extracellular matrix is ​​arranged between the above cells. The above-mentioned fragmented extracellular matrix is ​​a biocompatible material that consists of molecules identical to those present in living organisms. A three-dimensional tissue with a Young's modulus of 20 kPa or higher.

[28] The three-dimensional tissue according to

[27] , wherein the fragmented extracellular matrix is ​​a polypeptide having an RGD sequence.

[29] The three-dimensional tissue according to

[27] or

[28] , wherein the content of fragmented extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less is 5% by weight or more and 90% by weight or less based on the total weight of the three-dimensional tissue.

[30] A three-dimensional tissue according to any one of

[27] to

[29] , further comprising fragmented extracellular matrix with an average diameter greater than 1000 nm.

[31] A three-dimensional tissue according to any one of

[27] to

[29] , further comprising fragmented extracellular matrix having an average diameter of 1.5 μm or more and 8.5 μm or less.

[32] A three-dimensional tissue according to any one of

[27] to

[31] , wherein the ratio of the fragmented extracellular matrix with a smaller average diameter to the fragmented extracellular matrix with a larger average diameter in the three-dimensional tissue is 1:100 to 100:1 on a weight basis.

[33] A three-dimensional structure described in any of

[27] to

[32] , wherein the Young's modulus is 20 kPa to 250 kPa. [Effects of the Invention]

[0010] According to the present invention, a method for controlling the Young's modulus of a three-dimensional tissue mass manufactured using a highly biocompatible scaffolding material can be provided. Furthermore, a method for manufacturing a three-dimensional tissue mass having a desired Young's modulus can be provided. In addition, according to the present invention, a three-dimensional tissue mass with a Young's modulus of 20 kPa or higher can be provided.

[0011] According to the present invention, a three-dimensional tissue with a desired Young's modulus can be obtained, making it possible to create various tissue models close to the Young's modulus of living organisms and use them for drug evaluation and screening. Furthermore, according to the present invention, it is possible to obtain three-dimensional tissues with a high Young's modulus, which were previously difficult to manufacture using highly biocompatible scaffolding materials, making it possible to create cancer tissue models with a high Young's modulus in living organisms and use them for drug evaluation and screening. [Brief explanation of the drawing]

[0012] [Figure 1] This graph shows the fiber diameter distribution of fragmented collagen in the collagen dispersion of Example 2. [Figure 2] This graph shows the fiber diameter distribution of fragmented collagen in the collagen solution of Example 2. [Figure 3] This graph shows the change in Young's modulus as a function of CNF concentration in the CNF gel in Example 3. [Figure 4] This graph shows the results of comparing the Young's moduli of the CNF gel and CMF gel in Example 4. [Figure 5]This graph shows the results of comparing the Young's modulus of the three-dimensional tissue in Example 5. [Modes for carrying out the invention]

[0013] The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0014] <Method for controlling the Young's modulus of three-dimensional tissues> The method according to this embodiment controls the Young's modulus of a three-dimensional tissue including cells and extracellular matrix by adjusting the average diameter of the extracellular matrix.

[0015] In this specification, "three-dimensional tissue" means an aggregate of cells in which cells are arranged three-dimensionally via an extracellular matrix, and is an aggregate artificially created by cell culture. Therefore, a three-dimensional tissue includes cells and an extracellular matrix. An extracellular matrix may be arranged between at least some cells. There may be parts in the three-dimensional tissue where cells are in direct contact with each other. Preferably, the cells and extracellular matrix are distributed three-dimensionally in the three-dimensional tissue, with the extracellular matrix arranged between the cells, and more preferably, the cells and extracellular matrix are uniformly distributed three-dimensionally, with the extracellular matrix arranged between the cells. There are no particular restrictions on the shape of the three-dimensional tissue, and examples include sheet-like, spherical, nearly spherical, ellipsoidal, nearly ellipsoidal, hemispherical, nearly hemispherical, semicircular, nearly semicircular, rectangular prism-like, nearly rectangular prism, etc. Here, living tissue includes blood vessels, sweat glands, lymphatic vessels, sebaceous glands, etc., and its structure is more complex than that of a three-dimensional tissue. Therefore, three-dimensional tissue and living tissue can be easily distinguished. Furthermore, the three-dimensional tissue may be a mass assembled in a state of adhesion to the support, or it may be a mass assembled in a state of non-adhesion to the support.

[0016] The cells may be somatic cells or germ cells. Furthermore, the cells may be stem cells, or cultured cells such as primary cultured cells, passaged cultured cells, and cell line cells. In this specification, "stem cells" means cells that have the ability to self-renew and multipotency. Stem cells include pluripotent stem cells that have the ability to differentiate into any cell tumor, and tissue stem cells (also called somatic stem cells) that have the ability to differentiate into specific cell tumors. Examples of pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells). Examples of tissue stem cells include mesenchymal stem cells (e.g., bone marrow-derived stem cells), hematopoietic stem cells, and neural stem cells.

[0017] The cells constituting the three-dimensional tissue may include one or more types of cells. Examples of cells include fibroblasts (e.g., human skin fibroblasts (NHDF), human cardiac fibroblasts (NHCF), and human gingival fibroblasts (HGF)), mesenchymal cells such as chondrocytes and osteoblasts, vascular endothelial cells (e.g., human umbilical vein endothelial cells (HUVEC)), cancer cells such as colorectal cancer cells (e.g., human colorectal cancer cells (HT29)) and liver cancer cells, cardiomyocytes (e.g., human iPS cell-derived cardiomyocytes (iPS-CM)), epithelial cells (e.g., human gingival epithelial cells), keratinocytes, lymphatic endothelial cells, nerve cells, hepatocytes, tissue stem cells, embryonic stem cells, induced pluripotent stem cells, adherent cells (e.g., immune cells), smooth muscle cells (e.g., aortic smooth muscle cells (Arota-SMC)), and the like.

[0018] The total number of cells constituting the three-dimensional tissue according to this embodiment is not particularly limited and can be appropriately determined considering the thickness and shape of the three-dimensional tissue to be constructed, the size of the cell culture vessel used for construction, etc.

[0019] The extracellular matrix is ​​a material that fills the gaps between at least some cells in a three-dimensional tissue. The extracellular matrix may be an aggregate of extracellular matrix molecules, formed by multiple extracellular matrix molecules. Extracellular matrix molecules may be substances that exist outside cells in living organisms. Preferably, the extracellular matrix is ​​a biocompatible material that is identical to molecules that exist in living organisms. Any substance can be used as the extracellular matrix molecule, as long as it does not adversely affect cell growth and the formation of cell aggregates. Examples of extracellular matrix molecules include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, enteractin, fibrillin, and proteoglycans. The extracellular matrix may be used individually or in combination. The extracellular matrix may contain, for example, collagen, or may be collagen. When the extracellular matrix is ​​collagen, the collagen functions as a scaffold for cell adhesion, further promoting the formation of three-dimensional cell structures. In this embodiment, the extracellular matrix is ​​preferably a substance present outside animal cells, i.e., the extracellular matrix of an animal. The extracellular matrix molecule may be a modified or variant of the above-mentioned extracellular matrix molecule, or a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect cell growth and cell aggregate formation.

[0020] The extracellular matrix may have repeating sequences represented by Gly-XY, which are characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. Multiple Gly-XY sequences may be identical or different. Having repeating sequences represented by Gly-XY reduces constraints on the arrangement of molecular chains, resulting in even better function as a scaffolding material. In an extracellular matrix having repeating sequences represented by Gly-XY, the proportion of sequences represented by Gly-XY may be 80% or more of the total amino acid sequence, preferably 95% or more. The extracellular matrix may also be a polypeptide having an RGD sequence. An RGD sequence is a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). When the extracellular matrix has an RGD sequence, cell adhesion is further promoted, making it even more suitable as a scaffolding material. The extracellular matrix, which includes sequences represented by Gly-XY and RGD sequences, contains collagen, fibronectin, vitronectin, laminin, cadherin, and other components.

[0021] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. An example of non-fibrous collagen is type IV collagen.

[0022] Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0023] The extracellular matrix may contain at least one selected from the group consisting of collagen, laminin, and fibronectin, and preferably contains collagen. The collagen is preferably fibrous collagen, and more preferably type I collagen. Commercially available collagen may be used as the fibrous collagen, and specific examples include porcine skin-derived type I collagen and porcine skin-derived type I and type III collagen manufactured by Nippon Ham Co., Ltd. When a mixture of multiple types of collagen is used, such as type I and type III collagen, it may be separated into a single type of collagen by purification. For example, by adding salt to a solution containing a mixture of multiple types of collagen and separating the multiple types of collagen by centrifugation, and recovering the precipitate or supernatant, only the desired type of collagen can be obtained. The obtained desired type of collagen may be further dialysis to remove the salt, and after dialysis, it may be freeze-dried. The extracellular matrix may be an extracellular matrix derived from an animal. Examples of animal species from which the extracellular matrix may be derived include, but are not limited to, humans, pigs, and cattle. The extracellular matrix may use components derived from one type of animal, or it may use components derived from multiple types of animals in combination. The animal species from which the extracellular matrix is ​​derived may be the same as or different from the animal species from which the cells forming the three-dimensional organization originate.

[0024] Examples of extracellular matrix shapes include fibrous structures. Fibrous refers to a shape composed of thread-like extracellular matrix, or a shape composed of thread-like extracellular matrix molecules cross-linked. At least a portion of the extracellular matrix may be fibrous. The shape of the extracellular matrix is ​​the shape of a single mass of extracellular matrix (an aggregate of extracellular matrix) observed under a microscope, and the extracellular matrix preferably has the average diameter and / or average length described later. Fibrous extracellular matrix includes thin thread-like structures (fibers) formed by the aggregation of multiple thread-like extracellular matrix molecules, thread-like structures formed by further aggregation of fine fibers, and defibrations of these thread-like structures. When an extracellular matrix with a fibrous shape is included, the RGD sequence is preserved without being destroyed in the fibrous extracellular matrix, and it can function more effectively as a scaffold material for cell adhesion.

[0025] "Fragmentation" refers to reducing the size of aggregates of the extracellular matrix. Fragmented extracellular matrix (also called "fragmented extracellular matrix") may include defibrillated extracellular matrix. Defibrillated extracellular matrix is ​​obtained by defibrillating the extracellular matrix described above by applying physical force. For example, defibrillation is carried out under conditions that do not break the bonds within the extracellular matrix molecules.

[0026] The fragmented extracellular matrix may contain at least a portion of defibrated extracellular matrix. Alternatively, the fragmented extracellular matrix may consist solely of defibrated extracellular matrix. In other words, the fragmented extracellular matrix may be defibrated extracellular matrix. The defibrated extracellular matrix preferably contains defibrated collagen (defibrated collagen). The defibrated collagen preferably maintains the triple helix structure derived from collagen. The defibrated collagen may be a component that partially maintains the triple helix structure derived from collagen. By dispersing the fragmented collagen in an aqueous medium, it becomes easier for it to come into contact with cells in the aqueous medium, which can promote the formation of three-dimensional tissues.

[0027] The average length of the fragmented extracellular matrix may be between 100 nm and 400 μm, or between 100 nm and 200 μm. In one embodiment, from the viewpoint of facilitating the formation of thick tissue, the average length of the fragmented extracellular matrix may be between 5 μm and 400 μm, between 10 μm and 400 μm, between 22 μm and 400 μm, or between 100 μm and 400 μm. In another embodiment, from the viewpoint of facilitating stable tissue formation and improving redispersibility, the average length of the fragmented extracellular matrix may be 100 μm or less, 50 μm or less, 30 μm or less, 15 μm or less, 10 μm or less, 1 μm or less, or 100 nm or more. It is preferable that the average length of the majority of the fragmented extracellular matrix within the entire fragmented extracellular matrix falls within the above numerical range. Specifically, it is preferable that the average length of 95% of the fragmented extracellular matrix is ​​within the above numerical range. The fragmented extracellular matrix is ​​preferably fragmented collagen with an average length within the above range, and more preferably defibrillated collagen with an average length within the above range.

[0028] The average diameter of the fragmented extracellular matrix may be, for example, 20 nm to 30 μm, or 20 nm to 10 μm. In the method of this embodiment, the Young's modulus of the three-dimensional tissue can be increased by reducing the average diameter of the fragmented extracellular matrix contained in the three-dimensional tissue, and conversely, the Young's modulus of the three-dimensional tissue can be decreased by increasing the average diameter. Therefore, the method of this embodiment may include adjusting the average diameter of the extracellular matrix to the average diameter exemplified below.

[0029] Fragmented extracellular matrix with an average diameter of nano-order (less than 1000 nm) is also called nanofiber (NF). The average diameter of nanofibers may be, for example, 20 nm to 1000 nm, 20 nm to 500 nm, 20 nm to 200 nm, 40 nm to 130 nm, or 20 nm to 100 nm. On the other hand, fragmented extracellular matrix with an average diameter of micro-order (greater than 1000 nm) is also called microfiber (MF). The average diameter of microfibers may be, for example, greater than 1 μm and less than or equal to 30 μm, greater than 1 μm and less than or equal to 30 μm, greater than 1 μm and less than or equal to 10 μm, greater than or equal to 1.5 μm and less than or equal to 8.5 μm, or greater than or equal to 2 μm and less than or equal to 8.5 μm. In this embodiment, the shape of nanofibers and microfibers does not have to be fibrous, as long as they are the fragmented extracellular matrix described above.

[0030] The method of this embodiment may include adjusting all of the fragmented extracellular matrix contained in the three-dimensional tissue to nanofibers or microfibers. The method of this embodiment may also include adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters. For example, the three-dimensional tissue may be adjusted to contain both nanofibers and microfibers. Specifically, for example, the three-dimensional tissue may be adjusted to contain fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm and fragmented extracellular matrix with an average diameter of 1.5 μm to 8.5 μm. Alternatively, it may be adjusted to contain nanofibers of different average diameters. Specifically, for example, it may be adjusted to contain fragmented extracellular matrix with an average diameter of 20 nm to 100 nm and fragmented extracellular matrix with an average diameter of over 100 nm and up to 200 nm. By adjusting the weight-based ratio of fragmented extracellular matrix of different average diameters, a three-dimensional tissue with a desired Young's modulus can be obtained. For example, the fragmented extracellular matrix with a smaller average diameter (e.g., nanofibers) and the fragmented extracellular matrix with a larger average diameter (e.g., microfibers) may be used in a ratio of 1:100 to 100:1 by weight, or in a ratio of 1:10 to 10:1, or in a ratio of 1:3 to 3:1, or in a ratio of 1:2 to 2:1. Specifically, for example, fragmented extracellular matrices with an average diameter of 20 nm to 1000 nm and fragmented extracellular matrices with an average diameter of 1.5 μm to 8.5 μm can be used in a ratio of 1:100 to 100:1 by weight. Furthermore, the fragmented extracellular matrix with a smaller average diameter (e.g., nanofibers) may be used in a larger amount by weight than the fragmented extracellular matrix with a larger average diameter (e.g., microfibers). Specifically, for example, fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm may be used in greater quantities by weight than fragmented extracellular matrix with an average diameter of 1.5 μm to 8.5 μm.

[0031] The content of total fragmented extracellular matrix in a three-dimensional tissue may be, for example, 0.33% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, or 5% by weight or more, based on the total mass of fragmented extracellular matrix and cells. The content of fragmented extracellular matrix in a three-dimensional tissue may be, for example, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 5% by mass or less, or 1% by mass or less, based on the total mass of fragmented extracellular matrix and cells. The content of fragmented extracellular matrix in the three-dimensional tissue may be, for example, 0.33% to 90% by mass, 0.5% to 90% by mass, 1.0% to 90% by mass, 5% to 90% by mass, or 5% to 40% by mass, based on the total mass of the fragmented extracellular matrix and cells. In the method of this embodiment, the Young's modulus of the three-dimensional tissue may be controlled by adjusting the average diameter and content of the fragmented extracellular matrix. Therefore, the method of this embodiment may further include adjusting the content of fragmented extracellular matrix with different average diameters in the three-dimensional tissue. For example, the Young's modulus of the three-dimensional tissue can be increased by increasing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue or by decreasing the content of fragmented extracellular matrix with a large average diameter in the three-dimensional tissue. Conversely, the Young's modulus of a three-dimensional tissue can be reduced by decreasing the content of fragmented extracellular matrix with a small average diameter or by increasing the content of fragmented extracellular matrix with a large average diameter.In other words, adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters and adjusting the content of the fragmented extracellular matrix of different average diameters in the three-dimensional tissue may include increasing the content of fragmented extracellular matrix of smaller average diameters in the three-dimensional tissue (or decreasing the content of fragmented extracellular matrix of larger average diameters in the three-dimensional tissue) to increase the Young's modulus of the three-dimensional tissue, or decreasing the content of fragmented extracellular matrix of smaller average diameters in the three-dimensional tissue (or increasing the content of fragmented extracellular matrix of larger average diameters in the three-dimensional tissue) to decrease the Young's modulus of the three-dimensional tissue.

[0032] The above-mentioned ranges for average length and average diameter are optimized from the perspective of tissue formation; therefore, it is desirable that the average length or average diameter falls within the above range when used for tissue formation.

[0033] The average length and average diameter of fragmented extracellular matrix can be determined by measuring individual fragments of extracellular matrix using an optical microscope and performing image analysis. In this specification, "average length" refers to the average length of the measured sample in the longitudinal direction, and "average diameter" refers to the average length of the measured sample in the direction perpendicular to the longitudinal direction.

[0034] There are no particular restrictions on the method of fragmenting the extracellular matrix; it may be fragmented by applying physical force. Unlike enzymatic treatment, the molecular structure of extracellular matrix fragmented by applying physical force usually does not change from before fragmentation (the molecular structure is maintained). The method of fragmenting the extracellular matrix may be, for example, a method of finely crushing a clump of extracellular matrix. The extracellular matrix may be fragmented in a solid phase or in an aqueous medium. For example, the extracellular matrix may be fragmented by applying physical force using an ultrasonic homogenizer, agitator homogenizer, or high-pressure homogenizer. When using an agitator homogenizer, the extracellular matrix may be homogenized directly or in an aqueous medium such as physiological saline. Furthermore, the average diameter of the fragmented extracellular matrix can be adjusted by adjusting the homogenization time, number of times, etc. A method for producing nano-order fragmented extracellular matrix, particularly with a small average diameter (less than 1000 nm), includes, for example, homogenizing a dispersion containing extracellular matrix (e.g., collagen) in an aqueous medium, and then optionally incubating it in the aqueous medium at 10°C or below (e.g., 4°C ± 1°C). Examples of homogenization include using a homogenizer, stirring, applying pressure, etc., and stirring may also be done using a stirrer. The aqueous medium may be pure water or an aqueous solution containing phosphoric acid (e.g., PBS buffer). The incubation time at 10°C or below (e.g., 4°C ± 1°C) after homogenization may be, for example, 6 hours or more, 12 hours or more, 24 hours or more, 48 hours or more, or 72 hours or more. The aqueous medium used for homogenization (first aqueous medium) and the aqueous medium used for incubation (second aqueous medium) may be the same or different. The aqueous medium may or may not contain salt.

[0035] When fragmenting the extracellular matrix in an aqueous medium, the fragmented extracellular matrix can be produced, for example, by a method comprising the steps of fragmenting the extracellular matrix in an aqueous medium and removing the aqueous medium from the liquid containing the fragmented extracellular matrix and the aqueous medium (removal step). The removal step may be carried out, for example, by freeze-drying. "Removing the aqueous medium" does not mean that no moisture is attached to the fragmented extracellular matrix components, but rather that the amount of moisture attached is reduced to a level that can be reasonably achieved by the general drying methods described above.

[0036] At least a portion of the fragmented extracellular matrix may be cross-linked intermolecularly or intramolecularly. The fragmented extracellular matrix may be cross-linked within the molecules constituting the fragmented extracellular matrix, or cross-linked between the molecules constituting the fragmented extracellular matrix.

[0037] Methods for crosslinking include, for example, physical crosslinking by applying heat, ultraviolet light, or radiation, and chemical crosslinking by using crosslinking agents or enzymatic reactions, but the method is not particularly limited. Crosslinking (physical crosslinking and chemical crosslinking) may be crosslinking via covalent bonds.

[0038] In fragmented collagen, crosslinking may occur between collagen molecules (triple helix structure) or between collagen fibrils formed by collagen molecules. Crosslinking may be thermal crosslinking. Thermal crosslinking can be performed, for example, by heat treatment under reduced pressure using a vacuum pump. When thermal crosslinking of collagen molecules is performed, the defibrated collagen may be crosslinked by the formation of peptide bonds (-NH-CO-) between the amino groups of the collagen molecules or between the carboxyl groups of other collagen molecules.

[0039] Fragmented extracellular matrix can also be crosslinked using a crosslinking agent. The crosslinking agent may be, for example, one that can crosslink carboxyl groups with amino groups, or one that can crosslink amino groups with each other. From the viewpoint of economy, safety, and ease of handling, aldehyde-based, carbodiimide-based, epoxide-based, and imidazole-based crosslinking agents are preferred, for example. Specifically, water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide sulfonate can be used.

[0040] The degree of crosslinking can be appropriately selected depending on the type of fragmented extracellular matrix, the means of crosslinking, etc. The degree of crosslinking may be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, and may be 30% or less, 20% or less, or 15% or less.

[0041] When amino groups in extracellular matrix molecules are used for crosslinking, the degree of crosslinking can be quantified based on the TNBS method described in Non-Patent Document 2, etc. The degree of crosslinking obtained by the TNBS method may be within the range described above. The degree of crosslinking obtained by the TNBS method is the proportion of amino groups used for crosslinking out of the amino groups present in the extracellular matrix.

[0042] The degree of crosslinking may be calculated by quantifying the carboxyl groups. For example, in the case of an extracellular matrix insoluble in water, it may be quantified by the TBO (toluidine blue O) method. The degree of crosslinking obtained by the TBO method may be within the range described above.

[0043] The extracellular matrix content in a three-dimensional tissue may be 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more, based on the dry weight of the three-dimensional tissue, and may be 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, or 15% by mass or less. The extracellular matrix content in the three-dimensional tissue may be 0.01 to 90% by mass, 10 to 90% by mass, 10 to 80% by mass, 10 to 70% by mass, 10 to 60% by mass, 1 to 50% by mass, 10 to 50% by mass, 10 to 30% by mass, or 20 to 30% by mass, based on the dry weight of the three-dimensional tissue.

[0044] Here, "extracellular matrix in a three-dimensional tissue" refers to the extracellular matrix that constitutes the three-dimensional tissue, and may originate from either endogenous or exogenous extracellular matrix.

[0045] "Endogenous extracellular matrix" refers to the extracellular matrix produced by extracellular matrix-producing cells. Examples of extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, as mentioned above. The endogenous extracellular matrix may be fibrous or non-fibrous.

[0046] "Exogenous extracellular matrix" refers to extracellular matrix supplied from an external source. The three-dimensional tissue according to this embodiment includes fragmented extracellular matrix, which is exogenous extracellular matrix. The exogenous extracellular matrix may originate from the same or different animal species as the endogenous extracellular matrix. Examples of originating animal species include humans, pigs, and cattle. Furthermore, the exogenous extracellular matrix may be an artificial extracellular matrix.

[0047] When the extracellular matrix is ​​collagen, the exogenous extracellular matrix is ​​also called "exogenous collagen." Exogenous collagen, which refers to collagen supplied from outside, is an aggregate of collagen molecules formed by multiple collagen molecules, and specifically includes fibrous collagen and non-fibrous collagen. It is preferable that the exogenous collagen be fibrous collagen. The above-mentioned fibrous collagen refers to collagen that is the main component of collagen fibers, and examples include type I collagen, type II collagen, and type III collagen. The above-mentioned fibrous collagen may be commercially available collagen, and a specific example is type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. An example of exogenous non-fibrous collagen is type IV collagen.

[0048] In exogenous extracellular matrix, the animal species from which it originates may differ from that of the cells. Furthermore, if the cells include extracellular matrix-producing cells, the animal species from which the exogenous extracellular matrix originates may differ from that of the extracellular matrix-producing cells. In other words, the exogenous extracellular matrix may be a heterogeneous extracellular matrix.

[0049] In other words, when a three-dimensional tissue contains endogenous extracellular matrix and fragmented extracellular matrix, the extracellular matrix content of the three-dimensional tissue refers to the total amount of endogenous extracellular matrix and fragmented extracellular matrix. The extracellular matrix content can be calculated from the volume of the obtained three-dimensional tissue and the mass of the decellularized three-dimensional tissue.

[0050] For example, if the extracellular matrix contained in a three-dimensional tissue is collagen, one method for quantifying the amount of collagen in the three-dimensional tissue is to quantify hydroxyproline, as follows: Mix hydrochloric acid (HCl) with the lysate containing the three-dimensional tissue, incubate at a high temperature for a predetermined time, return to room temperature, and prepare a sample by diluting the supernatant obtained by centrifugation to a predetermined concentration. Prepare a standard by processing the hydroxyproline standard solution in the same way as the sample and then diluting it stepwise. Treat the sample and standard with hydroxyproline assay buffer and detection reagent as prescribed, and measure the absorbance at 570 nm. Calculate the amount of collagen by comparing the absorbance of the sample with that of the standard. Alternatively, the three-dimensional tissue may be directly suspended and dissolved in high-concentration hydrochloric acid, the lysate may be centrifuged, and the supernatant collected and used for collagen component quantification. Furthermore, the three-dimensional tissue to be dissolved may be in the state as recovered from the culture medium, or it may be dried after recovery to remove liquid components before dissolution. However, when quantifying collagen components by dissolving a three-dimensional tissue in its as-recovered state from the culture medium, the measured weight of the three-dimensional tissue is expected to vary due to the influence of culture medium components absorbed by the three-dimensional tissue and residual culture medium due to problems with the experimental procedure. Therefore, from the viewpoint of stably measuring the weight of the tissue and the amount of collagen per unit weight, it is preferable to use the weight after drying as the basis.

[0051] More specifically, the following methods can be used to quantify the amount of collagen.

[0052] (Sample preparation) The entire volume of the freeze-dried three-dimensional tissue is mixed with 6 mol / L HCl and incubated in a heat block at 95°C for at least 20 hours, then allowed to return to room temperature. After centrifugation at 13000 g for 10 minutes, the supernatant of the sample solution is collected. After diluting with 6 mol / L HCl as appropriate so that the results fall within the calibration curve in the measurement described later, 200 μL is diluted with 100 μL of ultrapure water to prepare the sample. 35 μL of the sample is used.

[0053] (Standard preparation) Add 125 μL of standard solution (1200 μg / mL in acetic acid) and 125 μL of 12 mol / L HCl to a screw-cap tube and mix. Incubate at 95°C for 20 hours on a heat block, then return to room temperature. Centrifuge at 13000 g for 10 minutes, then dilute the supernatant with ultrapure water to prepare S1 (300 μg / mL). Dilute S1 stepwise to prepare S2 (200 μg / mL), S3 (100 μg / mL), S4 (50 μg / mL), S5 (25 μg / mL), S6 (12.5 μg / mL), and S7 (6.25 μg / mL). Prepare S8 (0 μg / mL) using only 90 μL of 4 mol / L HCl.

[0054] (assay) Add 35 μL each of the standard and sample to a plate (QuickZyme Total Collagen Assay Kit, QuickZyme Biosciences). Add 75 μL of assay buffer (included in the kit) to each well. Seal the plate and incubate at room temperature for 20 minutes while shaking. Remove the seal and add 75 μL of detection reagent (reagent A:B = 30 μL:45 μL, included in the kit) to each well. Seal the plate, mix the solutions by shaking, and incubate at 60°C for 60 minutes. Cool thoroughly on ice, remove the seal, and measure the absorbance at 570 nm. Calculate the amount of collagen by comparing the absorbance of the sample with that of the standard.

[0055] The amount of collagen in a three-dimensional tissue can be defined by its area ratio or volume ratio. "Defining by area ratio or volume ratio" means, for example, making the collagen in the three-dimensional tissue distinguishable from other tissue components using known staining methods (e.g., immunohistochemical staining using anti-collagen antibodies, or Masson's trichrome staining), and then calculating the ratio of the collagen-containing area to the entire three-dimensional tissue using macroscopic observation, various microscopes, and image analysis software. When defining by area ratio, there are no limitations on which cross-section or surface of the three-dimensional tissue is used to define the area ratio; however, if the three-dimensional tissue is spherical, for example, it may be defined by a cross-sectional view passing through its approximate center.

[0056] For example, when defining collagen in a three-dimensional tissue by area ratio, the area ratio is preferably 0.01 to 99%, 1 to 99%, 5 to 90%, 7 to 90%, 20 to 90%, and more preferably 50 to 90%, based on the total area of ​​the three-dimensional tissue. The definition of "collagen in a three-dimensional tissue" is as described above. The area ratio of collagen constituting the three-dimensional tissue refers to the combined area ratio of endogenous collagen and exogenous collagen. The area ratio of collagen can be calculated, for example, by staining the obtained three-dimensional tissue with Masson's trichrome and determining the ratio of the area of ​​blue-stained collagen to the total area of ​​a cross-section passing through approximately the center of the three-dimensional tissue.

[0057] The three-dimensional tissue is preferably retained at a concentration of 0.25% trypsin, at a temperature of 37°C, pH 7.4, and reaction time of 15 minutes, with a retention rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more. Such a three-dimensional tissue is stable and less susceptible to enzymatic degradation during or after culture. The above retention rate can be calculated, for example, from the mass of the three-dimensional tissue before and after trypsin treatment.

[0058] The above three-dimensional tissue may have a residual rate of 70% or more after collagenase treatment at a collagenase concentration of 0.25%, a temperature of 37°C, a pH of 7.4, and a reaction time of 15 minutes, more preferably 80% or more, and even more preferably 90% or more. Such a three-dimensional tissue is stable and less susceptible to enzymatic degradation during or after culture.

[0059] The thickness of the three-dimensional tissue is preferably 10 μm or more, more preferably 100 μm or more, and even more preferably 1000 μm or more. Such a three-dimensional tissue has a structure closer to living tissue and is suitable as a substitute for experimental animals and as a transplant material. There is no particular upper limit to the thickness of the three-dimensional tissue, but for example it may be 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0060] Here, "thickness of the three-dimensional structure" refers to the distance between the two ends in a direction perpendicular to the main surface, if the three-dimensional structure is in the form of a sheet or a rectangular parallelepiped. If the main surface has irregularities, the thickness refers to the distance at the thinnest part of the main surface.

[0061] Furthermore, if the three-dimensional structure is spherical or nearly spherical, it refers to its diameter. Moreover, if the three-dimensional structure is ellipsoidal or nearly ellipsoidal, it refers to its minor axis. If the three-dimensional structure is nearly spherical or nearly ellipsoidal and has irregularities on its surface, the thickness refers to the shortest distance between two points where a line passing through the centroid of the three-dimensional structure intersects the surface.

[0062] The Young's modulus of the three-dimensional structure may be at 10°C to 45°C, preferably at 15°C to 40°C, more preferably at 20°C to 37°C, and even more preferably at 25°C (±1°C).

[0063] According to the method of this embodiment, a three-dimensional structure with a desired Young's modulus can be obtained. Therefore, the Young's modulus of the three-dimensional structure is not particularly limited, but may be, for example, 20kPa to 300kPa, 20kPa to 250kPa, 30kPa to 150kPa, 40kPa to 100kPa, 50kPa to 80kPa, or 50kPa to 70kPa. Furthermore, although the Young's modulus of the three-dimensional structure is not particularly limited, it may be, for example, 150kPa or more, 200kPa or more, or 250kPa or more.

[0064] The Young's modulus of a three-dimensional tissue can be measured using the EZ-test compact benchtop tester (manufactured by Shimadzu Corporation). Specifically, a gel (or three-dimensional tissue) placed in a typical culture vessel is subjected to a test using a rod-shaped jig (tip area: 11 mm²) set in the tester's load cell. 2 Compression is performed at a temperature of 25°C and a compression rate of 1.0 mm / min to obtain a stress-strain curve. Young's modulus (kPa) can be calculated from the slope of the elastic deformation region in the initial stage of stress rise in the obtained stress-strain curve.

[0065] <Method for manufacturing a three-dimensional tissue> The method for producing a three-dimensional tissue according to this embodiment includes the steps of: preparing a fragmented extracellular matrix whose average diameter is adjusted to match the desired Young's modulus of the three-dimensional tissue; mixing the extracellular matrix with cells in an aqueous medium to obtain a cell suspension; and incubating the cell suspension.

[0066] (Steps to prepare fragmented extracellular matrix) The manufacturing method of this embodiment includes a step of preparing a fragmented extracellular matrix whose average diameter is adjusted to match the desired Young's modulus of the three-dimensional tissue.

[0067] The three-dimensional tissue, Young's modulus, fragmented extracellular matrix, and methods for adjusting their average diameter are as described above. The manufacturing method of this embodiment may include a step of adjusting the average diameter of the fragmented extracellular matrix.

[0068] The average diameter of the fragmented extracellular matrix may be, for example, 20 nm to 30 μm, or 20 nm to 10 μm. In the manufacturing method of this embodiment, the Young's modulus of the three-dimensional tissue can be increased by reducing the average diameter of the fragmented extracellular matrix contained in the three-dimensional tissue, and conversely, the Young's modulus of the three-dimensional tissue can be decreased by increasing the average diameter. Therefore, the fragmented extracellular matrix with adjusted average diameter in the manufacturing method of this embodiment may include those adjusted to the average diameters exemplified below.

[0069] The fragmented extracellular matrix with adjusted average diameter may be nanofibers. The average diameter of the nanofibers may be, for example, 20 nm to 1000 nm, 20 nm to 500 nm, 20 nm to 200 nm, 40 nm to 130 nm, or 20 nm to 100 nm. Alternatively, the fragmented extracellular matrix with adjusted average diameter may be microfibers. The average diameter of the microfibers may be, for example, greater than 1 μm and less than or equal to 30 μm, greater than 1 μm and less than or equal to 30 μm, greater than 1 μm and less than or equal to 10 μm, greater than or equal to 1.5 μm and less than or equal to 8.5 μm, or greater than or equal to 2 μm and less than or equal to 8.5 μm.

[0070] In the manufacturing method of this embodiment, the fragmented extracellular matrix with adjusted average diameter may all be nanofibers or microfibers. Furthermore, the fragmented extracellular matrix with adjusted average diameter may include fragmented extracellular matrix with different average diameters. For example, it may include both nanofibers and microfibers, specifically, for example, fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm and fragmented extracellular matrix with an average diameter of 1.5 μm to 8.5 μm. It may also include nanofibers with different average diameters, specifically, for example, fragmented extracellular matrix with an average diameter of 20 nm to 100 nm and fragmented extracellular matrix with an average diameter of more than 100 nm and less than or equal to 200 nm. By adjusting the weight-based ratio of fragmented extracellular matrix with different average diameters, a three-dimensional tissue with a desired Young's modulus can be obtained. For example, the fragmented extracellular matrix with a smaller average diameter (e.g., nanofibers) and the fragmented extracellular matrix with a larger average diameter (e.g., microfibers) may be used in a ratio of 1:100 to 100:1 by weight, or in a ratio of 1:10 to 10:1, or in a ratio of 1:3 to 3:1, or in a ratio of 1:2 to 2:1. Specifically, for example, fragmented extracellular matrices with an average diameter of 20 nm to 1000 nm and fragmented extracellular matrices with an average diameter of 1.5 μm to 8.5 μm can be used in a ratio of 1:100 to 100:1 by weight. Furthermore, the fragmented extracellular matrix with a smaller average diameter (e.g., nanofibers) may be used in a larger amount by weight than the fragmented extracellular matrix with a larger average diameter (e.g., microfibers). Specifically, for example, fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm may be used in greater quantities by weight than fragmented extracellular matrix with an average diameter of 1.5 μm to 8.5 μm.

[0071] In the manufacturing method of this embodiment, the Young's modulus of the three-dimensional tissue can be controlled by adjusting the average diameter and content of the fragmented extracellular matrix. Therefore, in the manufacturing method of this embodiment, the step of preparing the fragmented extracellular matrix may be a step of preparing a fragmented extracellular matrix whose average diameter and content have been adjusted to match the desired Young's modulus of the three-dimensional tissue. For example, the Young's modulus of the three-dimensional tissue can be increased by increasing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue, or by decreasing the content of fragmented extracellular matrix with a large average diameter in the three-dimensional tissue. Conversely, the Young's modulus of the three-dimensional tissue can be decreased by decreasing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue, or by increasing the content of fragmented extracellular matrix with a large average diameter in the three-dimensional tissue. In other words, adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters and adjusting the content of the fragmented extracellular matrix of different average diameters in the three-dimensional tissue may include increasing the content of the fragmented extracellular matrix of smaller average diameters in the three-dimensional tissue to increase the Young's modulus of the three-dimensional tissue, or decreasing the content of the fragmented extracellular matrix of smaller average diameters in the three-dimensional tissue to decrease the Young's modulus of the three-dimensional tissue. Furthermore, the manufacturing method of this embodiment may further include a step of mixing the prepared fragmented extracellular matrix of different average diameters. Cells may be added after mixing the fragmented extracellular matrix of different average diameters, or they may be mixed together with the fragmented extracellular matrix of different average diameters.

[0072] The content of the fragmented extracellular matrix used in the manufacturing method of this embodiment may be, for example, 0.33% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, or 5% by mass or more, based on the total mass of the fragmented extracellular matrix and cells. The content of the fragmented extracellular matrix in the three-dimensional tissue may be, for example, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 5% by mass or less, or 1% by mass or less, based on the total mass of the fragmented extracellular matrix and cells. The content of fragmented extracellular matrix in the three-dimensional tissue may be, for example, 0.33% by mass or more and 90% by mass or less, 0.5% by mass or more and 90% by mass or less, 1.0% by mass or more and 90% by mass or less, 5% by weight or more and 90% by weight or less, or 5% by weight or more and 40% by weight or less, based on the total mass of fragmented extracellular matrix and cells.

[0073] Fragmented extracellular matrix can be obtained by the method described above. Fragmented extracellular matrix may also be obtained by fragmenting the extracellular matrix in an aqueous medium. That is, the manufacturing method according to this embodiment may include a step of fragmenting the extracellular matrix in an aqueous medium (fragmentation step) in the step of preparing the fragmented extracellular matrix.

[0074] The fragmented extracellular matrix may be as illustrated above and may contain fragmented collagen.

[0075] The manufacturing method according to this embodiment may further include a step of heating the extracellular matrix before the fragmentation step to crosslink at least a portion of the extracellular matrix, or a step of heating the extracellular matrix after the fragmentation step and before obtaining the cell suspension to crosslink at least a portion of the extracellular matrix. In the step of crosslinking at least a portion of the extracellular matrix, the lyophilized extracellular matrix may be heated.

[0076] In the crosslinking process, the temperature (heating temperature) and time (heating time) for heating the extracellular matrix can be determined as appropriate. The heating temperature may be, for example, 100°C or higher, 200°C or lower, or 220°C or lower. Specifically, the heating temperature may be, for example, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 220°C, etc. The heating time (the time to maintain the above heating temperature) can be set as appropriate depending on the heating temperature. For example, when heating at 100°C to 200°C, the heating time may be 6 hours or more and 72 hours or less, more preferably 24 hours or more and 48 hours or less. In the crosslinking process, heating may be carried out in the absence of a solvent, or under reduced pressure conditions.

[0077] The manufacturing method according to this embodiment may include a drying step after the fragmentation step in which the fragmented extracellular matrix is ​​dried.

[0078] In the drying process, the defibrated extracellular matrix is ​​dried. Drying may be carried out, for example, by freeze-drying. By performing the drying process after the defibration process, the aqueous medium is removed from the liquid containing the fragmented extracellular matrix components and the aqueous medium. The removal of the aqueous medium does not mean that there is absolutely no moisture attached to the fragmented extracellular matrix, but rather that there is no moisture attached to a degree that can be reasonably achieved by the general drying method described above.

[0079] (Step to obtain cell suspension) The manufacturing method of this embodiment includes the step of mixing the fragmented extracellular matrix with cells in an aqueous medium to obtain a cell suspension.

[0080] By dispersing fragmented extracellular matrix in an aqueous medium, it becomes easier for cells to come into contact with the matrix in the aqueous medium, which can promote the formation of three-dimensional tissues.

[0081] Methods for mixing fragmented extracellular matrix with cells in an aqueous medium include, but are not limited to, methods such as mixing an aqueous medium containing fragmented extracellular matrix with an aqueous medium containing cells, adding cells to an aqueous medium containing fragmented extracellular matrix, adding an aqueous medium containing extracellular matrix to a culture medium containing cells, adding cells to an aqueous medium containing fragmented extracellular matrix, or adding extracellular matrix and cells separately to a pre-prepared aqueous medium. Furthermore, the fragmented extracellular matrix and cells may be agitated in the aqueous medium by pipetting or other means.

[0082] In a method for adding cells to an aqueous medium containing fragmented extracellular matrix, the aqueous medium containing the fragmented extracellular matrix can be prepared by dispersing the fragmented extracellular matrix in an aqueous medium. The aqueous medium of the cell culture composition may be the same as or different from the aqueous medium used when mixing the extracellular matrix with the cells. If the aqueous medium is the same, a cell suspension may be obtained by mixing the cells with the aqueous medium containing the fragmented extracellular matrix. If the aqueous medium is different, the aqueous medium in the aqueous medium containing the fragmented extracellular matrix may be called the first aqueous medium, and the aqueous medium used when mixing with the cells may be called the second aqueous medium.

[0083] A cell suspension is obtained by dispersing fragmented extracellular matrix and cells in an aqueous medium. The aqueous medium may be a culture medium. There are no particular restrictions on the culture medium, and a suitable medium can be selected depending on the type of cells to be cultured. Examples of culture media include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The culture medium may be a serum-added medium or a serum-free medium. The culture medium may be a mixed medium obtained by mixing two types of media.

[0084] The concentration of the extracellular matrix in the process of obtaining the cell suspension can be appropriately determined according to the Young's modulus, shape, thickness, and size of the culture vessel of the target three-dimensional tissue. For example, the concentration of the extracellular matrix in the aqueous medium in the process of obtaining the cell suspension may be 0.1 to 90% by mass or 1 to 30% by mass.

[0085] The amount of fragmented extracellular matrix in the process of obtaining the cell suspension is, for example, 1.0 × 10⁻⁶ 6 The amount relative to the cells may be 0.1-100 mg, 0.5-50 mg, 0.8-25 mg, 1.0-10 mg, 1.0-5.0 mg, 1.0-2.0 mg, or 1.0-1.8 mg, and may be 0.7 mg or more, 1.1 mg or more, 1.2 mg or more, 1.3 mg or more, or 1.4 mg or more, and may be 7.0 mg or less, 3.0 mg or less, 2.3 mg or less, 1.8 mg or less, 1.7 mg or less, 1.6 mg or less, or 1.5 mg or less.

[0086] In the process of obtaining a cell suspension, the mass ratio of fragmented extracellular matrix to cells (fragmented extracellular matrix / cell) is preferably 1 / 1 to 1000 / 1, more preferably 9 / 1 to 900 / 1, and even more preferably 10 / 1 to 500 / 1.

[0087] The process may include adding fibrinogen and / or thrombin in the step of obtaining a cell suspension, or after obtaining a cell suspension but before the incubation step. When both fibrinogen and thrombin are added, for example, they may be added simultaneously, or fibrinogen may be added first and then thrombin. The timing of adding fibrinogen and / or thrombin is not particularly limited; for example, they may be added to an aqueous medium containing cells and fragmented extracellular matrix, or fibrinogen may be added to an aqueous medium containing cells and extracellular matrix, followed by the addition of adipocytes, and then thrombin. Adding fibrinogen and / or thrombin can suppress shrinkage that may occur in the incubation step described later, making it easier to control the shape and size of the three-dimensional tissue. In addition, the suspension of cells and fragmented extracellular matrix can be gelled, making it easier to detach the suspension from the culture vessel (support) after dropping it onto the culture vessel. Furthermore, if the cells include mature adipocytes, the adipocytes may become suspended due to the influence of lipid droplets within the mature adipocytes, potentially resulting in a heterogeneous culture environment between adipocytes and other cells. However, by gelling the suspension, it becomes easier to maintain a uniform mixture of each cell and the extracellular matrix, and to keep the cells and extracellular matrix in close proximity.

[0088] The manufacturing method of this embodiment may include a step of applying an external force to the cell suspension after the step of obtaining the cell suspension and before the incubation step of the cell suspension to accumulate the cells and fragmented extracellular matrix. Performing such a step results in a more uniform distribution of fragmented extracellular matrix and cells in the three-dimensional tissue. There are no particular limitations on the specific method, but one example is a method of centrifuging the cell suspension containing the extracellular matrix and cells.

[0089] (Steps to incubate the cell suspension) The manufacturing method of this embodiment includes the step of incubating the cell suspension.

[0090] In the manufacturing method according to this embodiment, the incubation step is a step of incubating cells that have come into contact with fragmented extracellular matrix. By incubation, cells that have come into contact with fragmented extracellular matrix can be cultured. Therefore, this step may also be referred to as the "culture step" below.

[0091] There are no particular restrictions on the incubation method, and it can be carried out in a manner suitable for the type of cell. For example, the incubation temperature may be 20°C to 40°C or 30°C to 37°C. The pH of the cell suspension may be 6 to 8 or 7.2 to 7.4. The incubation time may be 1 day to 2 weeks or 1 week to 2 weeks.

[0092] The incubator (support) used when incubating the cell suspension is not particularly limited and may be, for example, a well insert, a low-adhesion plate, or a plate with a bottom shape such as U-shaped or V-shaped. The cells may be cultured while attached to the support, or without being attached to the support, or they may be separated from the support during culture. When culturing the cells without being attached to the support, or when separating them from the support during culture, it is preferable to use a plate with a bottom shape such as U-shaped or V-shaped that inhibits cell adhesion to the support, or a low-adhesion plate.

[0093] The cell density in the culture medium during the culture process can be appropriately determined according to the shape, thickness, and size of the culture vessel of the target three-dimensional tissue. For example, the cell density in the culture medium during the culture process can be 1 to 10 8 It may be cells / mL, 10 3 ~10 7 The cell density may be cells / mL. Furthermore, the cell density in the culture medium during the culture process may be the same as the cell density in the aqueous medium during the contact process.

[0094] The three-dimensional tissue produced by the manufacturing method according to this embodiment preferably has a shrinkage rate of 20% or less during culture, more preferably 15% or less, and even more preferably 10% or less. The above shrinkage rate can be calculated, for example, by the following formula. In the formula, L1 represents the length of the longest part of the three-dimensional tissue on day 1 after culture, and L3 represents the length of the corresponding part of the three-dimensional tissue on day 3 after culture.

[0095] Contraction rate (%) = {(L1 - L3) / L1} × 100 In the example above, the contraction rate is calculated from the three-dimensional tissue on day 1 after culturing and the cell tissue on day 3 after culturing. However, it may also be calculated from the three-dimensional tissue at any point during the culture period, including the end of the culture period. For example, it may be calculated from the three-dimensional tissue on day 1 after culturing and the three-dimensional tissue on day 2 after culturing, or from the three-dimensional tissue on day 1 after culturing and the three-dimensional tissue on day 5 after culturing, or from the three-dimensional tissue on day 1 after culturing and the three-dimensional tissue on day 8 after culturing.

[0096] After the above culture step (hereinafter also referred to as the "first culture step"; the initial contact step is also referred to as the "first contact step"), the process may include a step of further contacting cells (second contact step) and a step of culturing cells (second culture step). The cells used in the second contact step and the second culture step may be the same species as the cells used in the first contact step and the first culture step, or they may be different species. A two-layered three-dimensional tissue can be produced by the second contact step and the second culture step. Furthermore, by repeatedly including the contact step and the culture step, a multi-layered three-dimensional tissue can be produced, making it possible to create more complex tissues that are closer to living organisms.

[0097] The above culture step may include culturing the cells that have come into contact with the fragmented extracellular matrix in a state where they are not adhered to the support. This makes it possible to produce a three-dimensional tissue that is aggregated in clumps without being adhered to the support. If the cells that have come into contact with the fragmented extracellular matrix are adhered to the support, the above culture step may include separating the cells that have come into contact with the fragmented extracellular matrix from the support. If the cells that have come into contact with the fragmented extracellular matrix are not adhered to the support from the beginning in the above culture step, a three-dimensional tissue that is aggregated in clumps without being adhered to the support can be produced by culturing them in that state.

[0098] The method for separating cells that have come into contact with the fragmented extracellular matrix from the support is not particularly limited. For example, a low-adhesion support may be used, and cells may be separated from the support by adding culture medium; cells may be physically separated directly from the support using instruments or the like; cells may be separated from the support by applying vibration; or a support coated with a functional material that breaks the bond between the support and cells in response to stimuli such as heat and light may be used, and cells may be separated from the support by applying these stimuli. When separating cells from the support by adding culture medium, the culture medium may be one of the culture media exemplified above.

[0099] In the culture process, methods for culturing cells that have come into contact with the fragmented extracellular matrix without them being attached to a support from the beginning include, for example, a method in which a suspension containing cells that have come into contact with the fragmented extracellular matrix is ​​gelled and then gently dropped into a culture medium for culturing, and a method in which the shape of cells that have come into contact with the fragmented extracellular matrix is ​​fixed to some extent in a high-viscosity solvent, and then only the solvent is removed and the cells are transferred to a culture vessel.

[0100] In the culture process described above, the timing of separating the cells that have come into contact with the fragmented extracellular matrix from the support is not particularly limited. For example, it may be done 1 to 7 days after the start of culture, 1 to 24 hours after the start of culture, 1 to 60 minutes after the start of culture, 5 to 30 minutes after the start of culture, or 10 to 20 minutes after the start of culture.

[0101] The culture period after separating the cells in contact with the fragmented extracellular matrix from the support is not particularly limited and may be 1 day or more, 1 to 21 days, 3 to 14 days, or 7 to 14 days.

[0102] The Young's modulus of the three-dimensional structure produced by the manufacturing method of this embodiment may be at 10°C to 45°C, preferably at 15°C to 40°C, more preferably at 20°C to 37°C, and even more preferably at 25°C (±1°C).

[0103] According to the manufacturing method of this embodiment, a three-dimensional structure with a desired Young's modulus can be obtained. Therefore, the Young's modulus of the three-dimensional structure is not particularly limited, but may be, for example, 1 kPa or more, 5 kPa or more, 10 kPa or more, 20 kPa or more, 40 kPa or more, 50 kPa or more, 75 kPa or more, 100 kPa or more, 150 kPa or more, 20 kPa to 250 kPa, 30 kPa to 150 kPa, 40 kPa to 100 kPa, 50 kPa to 80 kPa, or 50 kPa to 70 kPa.

[0104] The specific embodiments of the manufacturing method of this embodiment can be applied without limitation to the specific embodiments described above.

[0105] <Three-dimensional organization> One embodiment of the present invention provides a three-dimensional tissue comprising cells and a fragmented extracellular matrix having an average diameter of 20 nm to 1000 nm, wherein the fragmented extracellular matrix is ​​arranged between at least some of the cells, the fragmented extracellular matrix is ​​a biocompatible material having molecules identical to those present in living organisms, and has a Young's modulus of 20 kPa or more.

[0106] The measurement of fragmented extracellular matrix and Young's modulus is as described above.

[0107] The three-dimensional structure of this embodiment has a Young's modulus of 20 kPa or more, and may be 40 kPa or more, 50 kPa or more, 75 kPa or more, 100 kPa or more, 150 kPa or more, 200 kPa or more, or 250 kPa or more. Furthermore, the Young's modulus may be between 10 kPa and 300 kPa, between 10 kPa and 250 kPa, between 20 kPa and 150 kPa, or between 30 kPa and 100 kPa.

[0108] The content of fragmented extracellular matrix may be, for example, 0.33% by mass or more and 90% by mass or less, 0.5% by mass or more and 90% by mass or less, 1.0% by mass or more and 90% by mass or less, 5% by weight or more and 90% by weight or less, or 5% by weight or more and 40% by weight or less, based on the total mass of the three-dimensional tissue.

[0109] The three-dimensional tissue of this embodiment may further contain a fragmented extracellular matrix having an average diameter greater than 1000 nm, for example, a fragmented extracellular matrix having an average diameter of 1.5 μm or more and 8.5 μm or less. When a fragmented extracellular matrix having an average diameter greater than 1000 nm (for example, a fragmented extracellular matrix having an average diameter of 1.5 μm or more and 8.5 μm or less) is included, the fragmented extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less and the fragmented extracellular matrix having an average diameter greater than 1000 nm (for example, a fragmented extracellular matrix having an average diameter of 1.5 μm or more and 8.5 μm or less) may be used in a weight ratio of 1:100 to 100:1.

[0110] The specific embodiments of the three-dimensional structure of this embodiment can be applied without limitation to the specific embodiments described above.

[0111] <Three-dimensional tissue-forming agent> One embodiment of the present invention provides a three-dimensional tissue-forming agent comprising a fragmented extracellular matrix having an average diameter of 20 nm to 1000 nm and an aqueous medium, wherein the Young's modulus of the three-dimensional tissue is 20 kPa or more.

[0112] The three-dimensional tissue, fragmented extracellular matrix, aqueous medium, and Young's modulus measurements are as described above.

[0113] The three-dimensional tissue-forming agent in this embodiment can be used to form three-dimensional tissues with a Young's modulus of 20 kPa or higher. Alternatively, the three-dimensional tissue-forming agent may be mixed with cells to obtain a cell suspension, and the cell suspension may be incubated to produce the three-dimensional tissue.

[0114] The Young's modulus of a three-dimensional tissue may be 1 kPa or more, 5 kPa or more, 10 kPa or more, 20 kPa or more, 40 kPa or more, 50 kPa or more, 75 kPa or more, 100 kPa or more, 150 kPa or more, 200 kPa or more, or 250 kPa or more. Furthermore, the Young's modulus may be between 10 kPa and 300 kPa, between 10 kPa and 250 kPa, between 20 kPa and 150 kPa, or between 30 kPa and 100 kPa.

[0115] The Young's modulus of the three-dimensional tissue-forming agent in this embodiment can be measured in the same way as the three-dimensional tissue, and may be 20 kPa or more, 40 kPa or more, 50 kPa or more, 75 kPa or more, 100 kPa or more, 200 kPa or more, or 250 kPa or more. Furthermore, the Young's modulus may be between 10 kPa and 300 kPa, between 10 kPa and 200 kPa, between 20 kPa and 150 kPa, or between 30 kPa and 100 kPa. Also, the Young's modulus may be at 10°C to 45°C, preferably between 15°C and 40°C, more preferably between 20°C and 37°C, and even more preferably between 25°C (±1°C).

[0116] By incorporating the aforementioned fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm into an aqueous medium, a cell culture composition with a Young's modulus of 20 kPa or higher at 25°C (±1°C) can be prepared. The method for preparing the fragmented extracellular matrix with an average diameter of 20 nm to 1000 nm is as described above.

[0117] The content of fragmented extracellular matrix may be, for example, 0.33% by mass or more and 90% by mass or less, 0.5% by mass or more and 90% by mass or less, 1.0% by mass or more and 90% by mass or less, 5% by weight or more and 90% by weight or less, or 5% by weight or more and 40% by weight or less, based on the total mass of the three-dimensional tissue-forming agent.

[0118] The specific embodiments of the forming agent in this embodiment can be applied without limitation to the specific embodiments described above. [Examples]

[0119] [Example 1: Purification of Type I Collagen] A 0.2% by weight collagen solution was prepared by adding 1 g of mixed type I and type III collagen derived from porcine skin, manufactured by Nippon Ham Co., Ltd., to 500 mL of ultrapure water and incubating at 4°C for 12 hours. Then, 0.45 M NaCl and 5 mM Tris-HCl were added to the collagen solution and incubated at 4°C for 12 hours, after which 1.2 M NaCl was added and incubated for another 12 hours at 4°C. After incubation, the solution was centrifuged at 10,000 rpm for 15 minutes, and the supernatant was collected to obtain a purified type I collagen solution.

[0120] The obtained type I collagen solution was dialyzed with ultrapure water for 7 days (molecular weight cutoff (MWCO): 15 kDa), and then freeze-dried for 3 days to obtain purified type I collagen.

[0121] [Example 2: Preparation of collagen microfibers and collagen nanofibers and evaluation of fiber diameter] 50 mg of purified type I collagen obtained in Example 1 was suspended in 5 mL of 1 × PBS (pH=7.4) and homogenized at room temperature for 6 minutes using a stirring homogenizer to obtain a dispersion containing fragmented collagen. Figure 1 is a graph showing the fiber diameter distribution of the fragmented collagen in the dispersion. The average diameter of the obtained fragmented collagen was 5.03 ± 3.11 μm (number of samples: 25). These fragmented collagens will also be referred to as collagen microfibers (CMFs) below.

[0122] Furthermore, when 50 mg of purified type I collagen obtained in Example 1 was suspended in 5 mL of 1 × PBS (pH=7.4), homogenized at room temperature for 6 minutes using a stirring homogenizer, and then incubated at 4°C for 3 days, the fragmented collagen became even finer. Figure 2 is a graph showing the fiber diameter distribution of the fragmented collagen in the solution. The average diameter of the obtained fragmented collagen was 84.4 ± 43.0 nm (number of samples: 25). These fragmented collagens will also be referred to as collagen nanofibers (CNF) below.

[0123] [Example 3: Preparation of gel using collagen nanofibers and evaluation of Young's modulus] Using the same method as in Example 2, a CNF-containing solution prepared at a predetermined concentration (0.1-3.0 wt%) was dropped into 24 transwells at a rate of 300 μL / well, and then incubated at 37°C for 3 hours to gel, obtaining a CNF gel with a thickness of approximately 8-9 mm.

[0124] For comparison, a 0.1% by weight collagen gel was prepared using a porcine skin-derived type I collagen acid solution manufactured by Nippi Corporation, based on the protocol provided by the manufacturer (Nippi gel).

[0125] The Young's modulus of each gel was measured at 25°C as follows:

[0126] Measurements were taken using the EZ-test compact desktop testing machine (manufactured by Shimadzu Corporation). Specifically, a gel loaded into a 24-well insert (manufactured by Corning) was subjected to a test using a rod-shaped jig (tip area: 11 mm²) set in the load cell of the testing machine. 2 Compression was performed at a temperature of 25°C and a compression rate of 1.0 mm / min, and a stress-strain curve was obtained. Young's modulus (kPa) was calculated from the slope of the elastic deformation region in the initial stage of stress rise in the obtained stress-strain curve.

[0127] The results are shown in Figure 3. It was demonstrated that increasing the concentration (weight %) of CNF can increase the Young's modulus of CNF. It was even possible to produce a gel with a Young's modulus 160 times higher than that of commercially available Nippi gel.

[0128] [Example 4: Gel preparation using collagen microfibers and comparison of Young's modulus] Using the same method as in Example 2, a CMF-containing solution prepared at a predetermined concentration (0.2-2.0 wt%) was dropped into 24 transwells at a rate of 300 μL / well. The solution was then incubated at 37°C for 3 hours to gel, yielding a CMF gel with a thickness of approximately 8-9 mm. The Young's modulus of this CMF gel at 25°C was compared with that of the CNF gel prepared in Example 3.

[0129] The results are shown in Figure 4. The CNF gel showed a higher Young's modulus at the same concentration compared to the CMF gel. Furthermore, the transmittance (500 nm) of the 1 wt% CNF gel was 0.12%, which was higher than the transmittance (500 nm) of the CMF gel at the same concentration (0.03%).

[0130] [Example 5: Fabrication of three-dimensional tissue using collagen microfibers and comparison of Young's modulus] Using the collagen microfibers (CMF) and collagen nanofibers (CNF) prepared in Example 2, a three-dimensional tissue was fabricated as follows.

[0131] First, CMF and / or CNF were dispersed in DMEM to prepare 2wt% CNF dispersion, 1wt% CNF dispersion, 0.5% CNF dispersion, 1.33wt% CNF + 0.66% CMF dispersion, and 0.66wt% CNF + 1.33% CMF dispersion.

[0132] Human colorectal adenocarcinoma cell line (HT29) (ATCC number: CCL-247), which had been subcultured in a 10 cm dish using a standard method, was detached from the 10 cm dish and collected. HT29 was collected at a concentration of 1 × 10⁶ cells per 400 μL. 6 The cells were suspended in each of the above dispersions to obtain a cell suspension.

[0133] 400 μL of cell suspension was seeded into deep-well plates (EVERGREEN, #222-8636-010). The cells were incubated at 37°C, and the Young's modulus of each three-dimensional tissue at 25°C one day after seeding was measured in the same manner as in Examples 3 and 4.

[0134] The results are shown in Figure 5. In Figure 5, (total 2mg), (total 4mg), and (total 8mg) indicate the total amount of fragmented collagen (CMF and / or CNF) contained in the formed three-dimensional tissue, respectively. The specific gravity of the cell suspension is calculated at 1mg per 1μL. In the graphs of Figure 5, the first to third graphs from the left show the results of culturing cell suspensions obtained by mixing CNF dispersion and HT29 so that the final CNF concentrations were 2wt%, 1wt%, and 0.5wt%, respectively. The fourth graph from the left shows the results of mixing 2wt% CNF dispersion and 2wt% CMF dispersion in volume ratios of 66.6% and 33.3%, respectively. That is, it shows the results of culturing cell suspensions containing CNF and CMF in a ratio of 66.6:33.3 (approximately 1.3wt%:approximately 0.7wt%). The fifth image from the left shows the result of mixing a 2wt% CNF dispersion and a 2wt% CMF dispersion in volume ratios of 33.3% and 66.6%, respectively. In other words, it shows the results of culturing a cell suspension containing CNF and CMF in a ratio of 33.3:66.6 (approximately 0.7wt%:approximately 1.3wt%).

[0135] This demonstrated that even with the same total amount of fragmented collagen, mixing fragmented collagen with different fiber diameters results in different stiffness (Young's modulus) of the resulting three-dimensional tissue depending on the mixing ratio. Specifically, when creating a three-dimensional tissue containing CNF and CMF, it was found that even with the same total amount of fragmented collagen, increasing the proportion of CNF with smaller fiber diameters improved the Young's modulus, while increasing the proportion of CMF with larger fiber diameters decreased the Young's modulus (comparison of the first, fourth, and fifth images from the left in Figure 5). It was also shown that including 2 wt% or more of CNF allows for the creation of a three-dimensional tissue with a Young's modulus of 200 kPa or more (first image from the left in Figure 5), and including approximately 1.3 wt% or more of CNF allows for the creation of a three-dimensional tissue with a Young's modulus of 100 kPa or more (fourth image from the left in Figure 5). Furthermore, the created three-dimensional tissues were characterized by a three-dimensional arrangement of collagen between cells and a uniform distribution of cells and collagen.

Claims

1. In the production of a three-dimensional tissue containing cells and extracellular matrix, this method controls the Young's modulus of the three-dimensional tissue by adjusting the average diameter of the fragmented extracellular matrix. Adjusting the average diameter of the fragmented extracellular matrix includes adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters. The fragmented extracellular matrix of different average diameters includes a fragmented extracellular matrix with an average diameter of 20 nm or more and 1000 nm or less, and a fragmented extracellular matrix with an average diameter of more than 1000 nm and 30 μm or less. A method wherein the fragmented extracellular matrix comprises fragmented collagen.

2. The method according to claim 1, further comprising adjusting the content of the fragmented extracellular matrix.

3. The method according to claim 1 or 2, wherein the fragmented extracellular matrix is ​​a polypeptide having an RGD sequence.

4. The method according to claim 3, wherein the fragmented extracellular matrix is ​​a biocompatible material that is identical to molecules present in living organisms.

5. The method according to claim 1, further comprising adjusting the content of the fragmented extracellular matrix of different average diameters in the three-dimensional tissue.

6. Adjusting the three-dimensional tissue to contain extracellular matrix fragments of different average diameters and adjusting the content of the extracellular matrix fragments of different average diameters in the three-dimensional tissue, By increasing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue, the Young's modulus of the three-dimensional tissue can be increased, or The method according to claim 5, comprising reducing the Young's modulus of a three-dimensional tissue by reducing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue.

7. The method according to any one of claims 1 to 6, wherein the fragmented extracellular matrix having an average diameter of more than 1000 nm and less than or equal to 30 μm is a fragmented extracellular matrix having an average diameter of 1.5 μm or more and less than or equal to 8.5 μm.

8. The method according to any one of claims 6 to 7, wherein the fragmented extracellular matrix with a smaller average diameter and the fragmented extracellular matrix with a larger average diameter are used in a ratio of 1:100 to 100:1 by weight.

9. The method according to any one of claims 1 to 8, wherein the Young's modulus of the three-dimensional tissue is 20 kPa to 250 kPa.

10. A process for preparing fragmented extracellular matrix with an average diameter adjusted to match the desired Young's modulus of a three-dimensional tissue. A step of obtaining a cell suspension by mixing the fragmented extracellular matrix with cells in an aqueous medium, The step includes incubating the cell suspension, The step of preparing the fragmented extracellular matrix includes adjusting the three-dimensional tissue to contain fragmented extracellular matrix of different average diameters, The fragmented extracellular matrix of different average diameters includes a fragmented extracellular matrix with an average diameter of 20 nm or more and 1000 nm or less, and a fragmented extracellular matrix with an average diameter of more than 1000 nm and 30 μm or less. The aforementioned fragmented extracellular matrix includes fragmented collagen. A method for manufacturing a three-dimensional tissue.

11. The method according to claim 10, wherein the step of preparing the fragmented extracellular matrix is ​​the step of preparing a fragmented extracellular matrix whose average diameter and content are adjusted to match a desired Young's modulus of a three-dimensional tissue.

12. The method according to claim 10 or 11, wherein the fragmented extracellular matrix is ​​a polypeptide having an RGD sequence.

13. The method according to claim 12, wherein the fragmented extracellular matrix is ​​a biocompatible material that is identical to molecules present in living organisms.

14. The method according to claim 10, further comprising adjusting the content of the fragmented extracellular matrix of different average diameters in the three-dimensional tissue.

15. Adjusting the three-dimensional tissue to contain extracellular matrix fragments of different average diameters and adjusting the content of the extracellular matrix fragments of different average diameters in the three-dimensional tissue, By increasing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue, the Young's modulus of the three-dimensional tissue can be increased, or The method according to claim 14, comprising reducing the Young's modulus of a three-dimensional tissue by reducing the content of fragmented extracellular matrix with a small average diameter in the three-dimensional tissue.

16. The method according to any one of claims 1 to 15, wherein the fragmented extracellular matrix of different average diameters includes a fragmented extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less, and a fragmented extracellular matrix having an average diameter of 1.5 μm or more and 8.5 μm or less.

17. The method according to any one of claims 14 to 16, wherein the fragmented extracellular matrix with a smaller average diameter and the fragmented extracellular matrix with a larger average diameter are used in a ratio of 1:100 to 100:1 by weight.

18. The method according to any one of claims 10 to 17, wherein the Young's modulus of the three-dimensional tissue is 20 kPa to 250 kPa.

19. It comprises cells and fragmented extracellular matrix having an average diameter of 20 nm to 1000 nm. It further contains fragmented extracellular matrix with an average diameter greater than 1000 nm and less than or equal to 30 μm. The fragmented extracellular matrix is ​​arranged between the cells. The aforementioned fragmented extracellular matrix is ​​a biocompatible material that consists of molecules identical to those present in living organisms. The Young's modulus is between 20 kPa and 300 kPa. The aforementioned fragmented extracellular matrix is ​​a three-dimensional tissue containing fragmented collagen.

20. The three-dimensional tissue according to claim 19, wherein the fragmented extracellular matrix is ​​a polypeptide having an RGD sequence.

21. A three-dimensional tissue according to claim 19 or 20, wherein the content of fragmented extracellular matrix having an average diameter of 20 nm or more and 1000 nm or less is 5% by weight or more and 90% by weight or less, based on the total weight of the three-dimensional tissue.

22. A three-dimensional tissue according to any one of claims 19 to 21, wherein the fragmented extracellular matrix having an average diameter of more than 1000 nm and less than or equal to 30 μm is a fragmented extracellular matrix having an average diameter of 1.5 μm or more and less than or equal to 8.5 μm.

23. A three-dimensional tissue according to any one of claims 19 to 22, wherein the ratio of the fragmented extracellular matrix with a smaller average diameter to the fragmented extracellular matrix with a larger average diameter in the three-dimensional tissue is 1:100 to 100:1 on a weight basis.

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