Three-dimensional tissue structure and method for producing the same

Abstract

To provide a three-dimensional tissue structure that suppresses the inhibition of both functional structural formation and cell proliferation, while sustaining its thickness over an extended period.SOLUTION: A three-dimensional tissue structure contains cells and a matrix containing biocompatible molecules. The matrix is resistant to decomposition due to a substance secreted by the cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a three-dimensional structure and a method for producing the same. [Background technology]

[0002] A three-dimensional tissue structure has a structure in which cells are arranged three-dimensionally, and is used as a biological tissue model. Various methods for producing a three-dimensional tissue structure have been studied so far (for example, Patent Documents 1 and 2). Patent Document 1 describes a method for easily producing a three-dimensional tissue with a controlled shape, and specifically describes a method for producing a three-dimensional tissue, which includes a culture step of culturing cells in a culture solution containing a fragmented extracellular matrix component, fibrin, and an aqueous medium. Patent Document 2 describes a method for producing a three-dimensional cellular tissue, which includes a step A of mixing cells with a cationic substance and an extracellular matrix component to obtain a mixture, a step B of collecting the cells from the obtained mixture and forming a cell aggregate on a substrate, and a step C of culturing the cells to obtain a three-dimensional cellular tissue. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2020 / 203579 [Patent Document 2] International Publication No. 2017 / 146124 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in conventional methods for producing a three-dimensional tissue structure, multiple types of cells are co-cultured, which causes problems such as the inability to form or maintain structural characteristics such as blood vessels and lymphatic vessels in the three-dimensional tissue structure, or the inability of some cells to proliferate, etc. Furthermore, when a thick three-dimensional tissue structure is produced by a method for co-culturing multiple types of cells, the thickness is not maintained for a long period of time because the thickness is inversely proportional to the culture period, making long-term culture difficult.

[0005] An object of the present invention is to provide a three-dimensional tissue structure in which inhibition of functional structure formation and inhibition of cell proliferation are reduced and the thickness is maintained for a long period of time. Another object of the present invention is to provide a method for producing such a three-dimensional tissue structure. [Means for solving the problem]

[0006] The present invention includes, for example, the following inventions. [1] a matrix containing cells and biocompatible molecules, The matrix is ​​resistant to degradation by substances secreted by the cells. [2] The three-dimensional tissue structure described in [1], wherein the matrix contains an inhibitor of degradation by a substance secreted by the cells. [3] The three-dimensional tissue structure according to [1] or [2], wherein the biocompatible molecule is resistant to degradation by a substance secreted by the cells. [4] The method comprises the steps of: (a) providing a first portion or portions each of which is made of the matrix; and (b) providing a second portion or portions each of which is made of a tissue structure containing the cells; The three-dimensional tissue structure according to any one of [1] to [3], wherein the second portions are arranged so as to be in contact with other second portions via the first portions. [5] The three-dimensional tissue structure according to any one of [1] to [4], wherein the matrix is ​​permeable to humoral factors but not permeable to the cells. [6] The three-dimensional tissue structure according to any one of [1] to [5], wherein the matrix is ​​in the form of one or more selected from the group consisting of hydrogel, hydrogel fiber, and hydrogel film. [7] The three-dimensional tissue structure according to any one of [1] to [6], which has a thickness of 600 μm or more. [8] The method includes the steps of mixing cells with a matrix precursor containing a biocompatible molecule in an aqueous medium, and then forming a matrix from the matrix precursor, The method for producing a three-dimensional tissue structure, wherein the matrix is ​​resistant to degradation by substances secreted by the cells. [9] The method according to [8], further comprising a step of culturing the cells in the formed matrix.

[10] The method includes the steps of: preparing a plurality of tissue structures containing cells; and forming a matrix between the plurality of tissue structures from a matrix precursor containing a biocompatible molecule; The method for producing a three-dimensional tissue structure, wherein the matrix is ​​resistant to degradation by substances secreted by the cells.

[11] The method for producing a three-dimensional tissue structure according to any one of [8] to

[10] , wherein the matrix contains an inhibitor of degradation by a substance secreted by the cells.

[12] The method for producing a three-dimensional tissue structure according to any one of [8] to

[11] , wherein the biocompatible molecule is resistant to degradation by a substance secreted by the cells.

[13] The method for producing a three-dimensional tissue structure according to any one of [8] to

[12] , wherein the matrix is ​​in the form of one or more selected from the group consisting of hydrogel, hydrogel fiber, and hydrogel film.

[14] a matrix containing biocompatible molecules; The matrix is ​​resistant to degradation by substances secreted by cells, in the unit for forming a three-dimensional tissue structure. Effect of the Invention

[0007] According to the present invention, it is possible to provide a three-dimensional tissue structure in which inhibition of functional structure formation and inhibition of cell proliferation are reduced and the thickness is maintained for a long period of time. The present invention can also provide a method for producing such a three-dimensional tissue structure. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram showing a cross section of a three-dimensional tissue structure according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0010] [Three-dimensional tissue structure] As used herein, a "biocompatible molecule" refers to a molecule that does not adversely affect cell growth, etc., and does not interfere with the formation of a three-dimensional tissue structure. A "matrix" refers to a structure in which a three-dimensional mesh structure is formed.

[0011] The three-dimensional tissue structure according to this embodiment includes cells and a matrix containing biocompatible molecules, the matrix being resistant to degradation by substances secreted by the cells.

[0012] The matrix is ​​resistant to degradation by substances secreted by cells, and therefore can maintain a three-dimensional mesh structure. This allows an appropriate distance to be maintained between cells or tissues containing cells, and allows the transfer of nutrients (e.g., culture medium, etc.), oxygen, waste products, etc., via the matrix. In addition, the loss of thickness associated with culture is reduced. This reduces inhibition of the formation of functional structures and inhibition of cell proliferation, resulting in a three-dimensional tissue structure whose thickness is maintained for a long period of time.

[0013] The three-dimensional tissue structure according to the present embodiment can be used, for example, as a biological tissue model or a solid cancer model, although it is not particularly limited thereto. Examples of biological tissue models include skin, heart, liver, stomach, small intestine, large intestine, vena cava, and aorta. Examples of solid cancer models include breast cancer, gastric cancer, colon cancer, esophageal cancer, liver cancer, ovarian cancer, prostate cancer, and pancreatic cancer.

[0014] The total number of cells constituting the three-dimensional tissue structure of this embodiment is not particularly limited, and is determined appropriately taking into consideration the thickness and shape of the three-dimensional tissue structure to be constructed, the size of the cell culture vessel used for construction, etc.

[0015] The three-dimensional tissue structure according to the present embodiment includes cells. The cells in the three-dimensional tissue structure may be, for example, mature somatic cells, or undifferentiated cells such as stem cells. Specific examples of somatic cells include, for example, nerve cells, dendritic cells, immune cells, vascular endothelial cells, lymphatic endothelial cells, fibroblasts, epithelial cells, cardiac myocytes, pancreatic islet cells, smooth muscle cells, bone cells, alveolar epithelial cells, and spleen cells. Examples of stem cells include ES cells, iPS cells, and mesenchymal stem cells. Other cells may be normal cells, or may be cells such as cancer cells in which any of the cell functions is enhanced or suppressed. A "cancer cell" is a cell that is derived from a somatic cell and has acquired infinite proliferation ability. Examples of cancer cells include breast cancer cells, gastric cancer cells, colon cancer cells, esophageal cancer cells, liver cancer cells, ovarian cancer cells, prostate cancer cells, and pancreatic cancer cells. The three-dimensional tissue structure may include one type of cell, or a combination of two or more types of cells.

[0016] The cells may include stromal cells or cancer stromal cells. Stromal cells are cells that constitute a supporting tissue for epithelial cells. Cancer stromal cells are cells that constitute a supporting tissue for cancer cells in tumor tissue. Stromal cells include fibroblasts, immune cells, pericytes, nerve cells, mast cells, epithelial cells, cardiomyocytes, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells, and the like. Cancer stromal cells include cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), cancer-associated (peripheral) endothelial (vascular) cells (CAEs), and the like.

[0017] The origin of the cells contained in the three-dimensional tissue structure is not particularly limited, and may be, for example, cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats.

[0018] A matrix containing a biocompatible molecule (hereinafter, simply referred to as "matrix") is resistant to degradation by substances secreted by the above-mentioned cells. Examples of biocompatible molecules include extracellular matrix components, fibrin, collagen, gelatin, hyaluronic acid, alginic acid, pectin, chitosan, cellulose, polylactic acid (PLA), polycaprolactone (PCL), hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), alginic acid, gelatin methacryloyl, and the like. The biocompatible molecule may consist of one of the above-mentioned molecules, or may contain a combination of two or more of them.

[0019] The form of the matrix may be, for example, hydrogel, hydrogel fiber, hydrogel film, etc. The form of the matrix contained in the three-dimensional tissue structure may be one type or two or more types. The form of the matrix contained in the three-dimensional tissue structure is preferably one or more types selected from the group consisting of hydrogel, hydrogel fiber, and hydrogel film, and is also preferably hydrogel and / or hydrogel fiber.

[0020] The term "hydrogel" refers to a three-dimensional network structure formed by crosslinking polymers through hydrogen bonds, ionic bonds, coordinate bonds, covalent bonds, etc., and containing liquid such as water inside the three-dimensional network structure. A preferred hydrogel is fibrin gel. Fibrin is a component that is generated when thrombin acts on fibrinogen to release A chains and B chains from the N-terminus of Aα chains and Bβ chains. Fibrin is formed by contacting fibrinogen with thrombin.

[0021] "Hydrogel fiber" refers to fibers formed by spinning a solution obtained by dissolving a hydrogel. The method for spinning the hydrogel solution is not particularly limited, but for example, it can be spun by electrospinning. When the matrix is ​​in the form of hydrogel fiber, the matrix may be a hydrogel fiber formed body. "Hydrogel fiber formed body" refers to something formed from hydrogel fiber. The hydrogel fiber formed body may be, for example, a nonwoven fabric, woven fabric, etc. formed from hydrogel fiber obtained by dissolving the above-mentioned hydrogel and spinning it.

[0022] The term "hydrogel film" refers to a film formed of a hydrogel. The method for producing the hydrogel film is not particularly limited, but for example, the hydrogel film can be produced by forming a film of the solution obtained by dissolving the hydrogel, and then removing the solvent.

[0023] "Resistant to degradation" may mean that the matrix shape can be maintained for, for example, 4 days or more when the matrix and cells are allowed to coexist. The matrix resistant to degradation by a substance secreted by cells may be, for example, a matrix further containing an inhibitor of degradation by a substance secreted by cells, or a matrix containing a biocompatible molecule resistant to degradation by a substance secreted by cells.

[0024] Among the substances secreted by cells, examples of substances capable of decomposing the matrix include enzymes such as hydrolases, etc. Examples of the enzymes include plasmin, proteases (e.g., serine proteases, collagenases, etc.), etc.

[0025] Examples of inhibitors of substances that degrade the matrix include protease inhibitors. Examples of protease inhibitors include α2-antiplasmin, serine protease (e.g., trypsin, chymotrypsin, elastase, etc.) inhibitors, cysteine ​​protease (e.g., papain, cathepsin, lysosomal cathepsin, etc.) inhibitors, aspartic acid protease (e.g., peptin, renin, etc.) inhibitors, metalloprotease (e.g., thermolysin, carboxypeptidase A, etc.) inhibitors, etc. In addition, the protease inhibitor may be, for example, a commercially available protease inhibitor cocktail (e.g., manufactured by Sigma, Roche, etc.).

[0026] The content of the inhibitor in the matrix may be, for example, 0.001% by mass or more, 0.005% by mass or more, 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, or 5% by mass or more, based on the mass of the matrix. The content of the inhibitor in the matrix may be, for example, 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.5% by mass or less, 0.1% by mass or less, 0.05% by mass or less, or 0.01% by mass or less, based on the mass of the matrix.

[0027] Examples of biocompatible molecules that are resistant to degradation by substances secreted by cells include the above-mentioned biocompatible molecules in which a site that is degraded by an enzyme has been modified.

[0028] The matrix may or may not contain cells. When the matrix contains cells, the cells may be the same type as the above-mentioned cells or may be different types. When the matrix contains cells, the cells may be one type or two or more types.

[0029] The matrix may be permeable to humoral factors (eg, nutrients, oxygen, waste products, etc.) but impermeable to cells.

[0030] A matrix that allows humoral factors (e.g., nutrients, oxygen, waste products, etc.) to pass through but does not allow cells to pass through may have a mesh size of 1 μm or more, 2 μm or more, 3 μm or more, 4 μm or more, or 5 μm or more. The mesh size of the matrix may be, for example, 15 μm or less, 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, or 10 μm or less.

[0031] The content of the biocompatible molecule in the matrix may be, for example, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more based on the mass of the matrix. The content of the biocompatible molecule in the matrix may be, for example, 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, or 50% by mass or less based on the mass of the matrix.

[0032] The three-dimensional tissue structure according to this embodiment may have one or more first parts made of the matrix and multiple second parts made of tissues containing the cells, and each of the second parts may be arranged so as to contact with another second part via the first part. Fig. 1 is a schematic diagram showing a cross section of a three-dimensional tissue structure according to one embodiment. Here, in this specification, the term "tissue" refers to a cell aggregate (a cluster of cells) in which cells are arranged three-dimensionally, and is an aggregate artificially produced by cell culture.

[0033] The three-dimensional tissue structure 100 shown in FIG. 1 has a first portion 10 and a plurality of second portions 20, and each of the second portions 20 is disposed so as to be in contact with the other second portions 20 via the first portion 10. Note that in FIG. 1, the first portion and the second portion are partially omitted. The first portion 10 is made of the matrix, and the second portion 20 is made of a tissue body containing cells. In this case, humoral factors (nutritional components, oxygen, waste products, etc.) can be transported via the first portion, so that inhibition of the formation of functional structures and inhibition of cell proliferation are further reduced. In addition, a three-dimensional tissue structure can be obtained that maintains its thickness even after long-term culture.

[0034] The first portions 10 and the second portions 20 may be arranged alternately in the direction of gravity (the Z-axis direction in FIG. 1), may be arranged alternately in the depth direction (the Y-axis direction in FIG. 1), or may be arranged alternately in the width direction (the X-axis direction in FIG. 1).

[0035] The first portion 10 is made of the matrix. In the three-dimensional tissue structure according to one embodiment, when there are a plurality of first portions, the number of first portions may be, for example, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more. In the three-dimensional tissue structure according to this embodiment, the number of first portions may be 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, or 20 or less.

[0036] The shape of the first portion 10 is not particularly limited, and examples thereof include a sphere, an approximately sphere, an ellipsoid, an approximately ellipsoid, a hemisphere, an approximately hemisphere, a semicircle, an approximately semicircle, a polyhedron, and the like. A polyhedron is preferable, and a rectangular parallelepiped or approximately rectangular parallelepiped is more preferable.

[0037] The first portion 10 may have a larger volume than the second portion, as shown by the portion surrounded by a dotted line in Fig. 1, or may be formed by contacting a plurality of first portions together. In the three-dimensional tissue structure 100, the first portions may be arranged so as to be in a straight line in any one or all of the directions of gravity, depth, and width. In this case, a tubular structure 30 made of the first portions 10 can be formed in the three-dimensional tissue structure 100. When the tubular structure 30 is formed in the three-dimensional tissue structure 100, the three-dimensional tissue structure has an even better permeability for humoral factors (nutritional components, oxygen, waste products, etc.).

[0038] The second portion 20 is made of a tissue containing cells, and the cells are as described above. The second portion 20 preferably does not contain the matrix. The tissue may contain an extracellular matrix component and / or a polyelectrolyte, which will be described later.

[0039] The tissue may contain a hydrogel. The hydrogel may or may not be resistant to degradation by a substance secreted by cells in the tissue. When the tissue contains a hydrogel, the tissue may be embedded in the hydrogel. The tissue being embedded in the hydrogel means that the hydrogel is present in at least some or all of the intercellular spaces outside the tissue or outside and inside the tissue.

[0040] In one embodiment, the tissue structure may have a configuration in which the outside of the tissue structure is covered with hydrogel, the inside of the tissue structure is densely packed with cells, and no hydrogel is present in the intercellular spaces inside the cell structure. By covering only the outside with hydrogel, the three-dimensional arrangement can be maintained and the three-dimensional structure can be maintained.

[0041] In the three-dimensional tissue structure according to one embodiment, the second portion may be, for example, 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more. In the three-dimensional tissue structure according to this embodiment, the second portion may be 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, or 20 or less.

[0042] The shape of the second portion 20 is not particularly limited, and examples thereof include a sphere, an approximately sphere, an ellipsoid, an approximately ellipsoid, a hemisphere, an approximately hemisphere, a semicircle, an approximately semicircle, a polyhedron, etc., with a polyhedron being preferred, and a rectangular parallelepiped or approximately rectangular parallelepiped being more preferred. The shape of the second portion may be the same as or different from the shape of the first portion 10.

[0043] In the three-dimensional tissue structure 100, the first portions 10 and the second portions 20 may be alternately bonded together. When the matrix form in the first portions 10 is hydrogel and / or hydrogel fiber, the first portions 10 and the second portions 20 can be bonded together by bringing the first portions 10 before gelation into contact with the second portions 20 and then gelling the first portions 10.

[0044] The three-dimensional tissue structure according to this embodiment may contain an extracellular matrix component. The extracellular matrix component may be disposed between at least some of the cells in the three-dimensional tissue structure.

[0045] As used herein, the term "extracellular matrix component" refers to an assembly of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. The term "extracellular matrix molecule" refers to a substance present outside cells in an organism. Any substance can be used as the extracellular matrix as long as it does not adversely affect cell growth and cell aggregate formation. Specific examples of extracellular matrix molecules include, but are not limited to, collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, tenascin, entactin, fibrillin, and cadherin. The extracellular matrix component may be used alone or in combination.

[0046] The extracellular matrix may be a modified or variant of the above-mentioned extracellular matrix, or may be a polypeptide such as a chemically synthesized peptide, so long as it does not adversely affect cell growth and cell aggregate formation. The extracellular matrix may have a repeat of a sequence represented by Gly-XY, which is characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. The multiple Gly-XYs may be the same or different. By having a repeat of a sequence represented by Gly-XY, the molecular chain arrangement is less restricted, and for example, the function as a scaffold material during cell culture is further improved. In the extracellular matrix having a repeat of a sequence represented by Gly-XY, the ratio of the sequence represented by Gly-XY may be 80% or more, preferably 95% or more, of the total amino acid sequence. The extracellular matrix may also be a polypeptide having an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). By having the RGD sequence, cell adhesion is further promoted, making it more suitable as a scaffold material for cell culture, for example. Examples of extracellular matrices containing a sequence represented by Gly-XY and an RGD sequence include collagen, fibronectin, vitronectin, laminin, cadherin, etc.

[0047] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen means collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. Examples of non-fibrous collagen include type IV collagen.

[0048] Proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0049] The shape of the extracellular matrix component may be, for example, fibrous. The term "fibrous" refers to a shape composed of thread-like extracellular matrix components, or a shape composed of thread-like extracellular matrix components cross-linked between molecules. At least a part of the extracellular matrix component may be fibrous. The shape of the extracellular matrix component is the shape of a mass of extracellular matrix components (aggregates of extracellular matrix components) observed under a microscope, and the extracellular matrix component preferably has an average diameter and / or average length as described below. The fibrous extracellular matrix component includes thin threads (fibers) formed by the aggregation of multiple thread-like extracellular matrix molecules, threads formed by further aggregation of filaments, and those obtained by defibrating these threads. When the extracellular matrix component has a fibrous shape, the RGD sequence is preserved without being destroyed in the fibrous extracellular matrix component, and the fibrous extracellular matrix component can function more effectively as a scaffold for cell adhesion.

[0050] The extracellular matrix component 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, more preferably type I collagen. The fibrous collagen may be commercially available collagen, and a specific example thereof is porcine skin-derived type I collagen manufactured by Nippon Ham Co., Ltd.

[0051] The extracellular matrix component may be an extracellular matrix component derived from an animal. Examples of animal species from which the extracellular matrix component is derived include, but are not limited to, humans, pigs, cows, etc. The extracellular matrix component may be derived from one type of animal, or may be derived from a combination of multiple types of animals.

[0052] The extracellular matrix component may include a fragmented extracellular matrix component. "Fragmentation" means breaking down an aggregate of extracellular matrix components into smaller sizes. The fragmented extracellular matrix component may include a defibrated extracellular matrix component. A defibrated extracellular matrix component is a component obtained by defibrating the above-mentioned extracellular matrix component by application of a physical force. For example, defibration is performed under conditions that do not break the bonds within the extracellular matrix molecules.

[0053] Fragmented extracellular matrix components can be produced, for example, by a method that includes a step of fragmenting an extracellular matrix component (fragmentation step).

[0054] The method for fragmenting the extracellular matrix components is not particularly limited, and the components may be fragmented by applying a physical force. Unlike enzyme treatment, the molecular structure of the fragmented extracellular matrix components by applying a physical force usually does not change from that before fragmentation (the molecular structure is maintained). The method for fragmenting the extracellular matrix components may be, for example, a method for finely breaking down a lump of extracellular matrix components. The extracellular matrix components may be fragmented in a solid phase or in an aqueous medium. For example, the extracellular matrix components may be fragmented by applying a physical force using an ultrasonic homogenizer, an agitation homogenizer, a high-pressure homogenizer, or the like. When using an agitation homogenizer, the extracellular matrix components may be homogenized as they are, or may be homogenized in an aqueous medium such as physiological saline. It is also possible to obtain millimeter-sized or nanometer-sized fragmented extracellular matrix components by adjusting the homogenization time, number of times, and the like. When the extracellular matrix components are fragmented in an aqueous medium, the fragmented extracellular matrix components can be produced, for example, by a method including a step of fragmenting the extracellular matrix components in an aqueous medium and a step of removing the aqueous medium from a liquid containing the fragmented extracellular matrix components and the aqueous medium (a removal step). The removal step may be carried out, for example, by a freeze-drying method. "Removing the aqueous medium" does not mean that no moisture is attached to the fragmented extracellular matrix components, but means that moisture is not attached to a degree that can be reasonably achieved by the above-mentioned general drying techniques.

[0055] The diameter and length of the fragmented extracellular matrix components can be determined by analyzing individual fragmented extracellular matrix components by electron microscopy.

[0056] The average length of the fragmented extracellular matrix components may be 100 nm or more and 400 μm or less, or 100 nm or more and 200 μm or less. In one embodiment, the average length of the fragmented extracellular matrix components may be 5 μm or more and 400 μm or less, 10 μm or more and 400 μm or less, or 100 μm or more and 400 μm or less, from the viewpoint of facilitating the formation of a thick three-dimensional tissue structure. In another embodiment, the average length of the fragmented extracellular matrix components 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 components among the entire fragmented extracellular matrix components is within the above numerical range. Specifically, of all the fragmented extracellular matrix components, it is preferable that the average length of 50% or more of the fragmented extracellular matrix components is within the above numerical range, and it is more preferable that the average length of 95% of the fragmented extracellular matrix components is within the above numerical range. The fragmented extracellular matrix components are preferably fragmented collagen components having an average length within the above numerical range.

[0057] The average diameter of the fragmented extracellular matrix component may be 50 nm to 30 μm, 4 μm to 30 μm, or 5 μm to 30 μm. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average diameter within the above range.

[0058] The average length and average diameter of the fragmented extracellular matrix components can be determined by measuring each fragmented extracellular matrix component using an optical microscope or the like and analyzing the images. In this specification, "average length" means the average value of the length in the longitudinal direction of the measured sample, and "average diameter" means the average value of the length in the direction perpendicular to the longitudinal direction of the measured sample.

[0059] The fragmented collagen component is also called "fragmented collagen component". The "fragmented collagen component" refers to a collagen component such as a fibrous collagen component that has been fragmented and that maintains a triple helix structure. The average length of the fragmented collagen component is preferably 100 nm to 200 μm, more preferably 22 μm to 200 μm, and even more preferably 100 μm to 200 μm. The average diameter of the fragmented collagen component is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0060] At least a portion of the extracellular matrix component may be intermolecularly or intramolecularly crosslinked. The extracellular matrix component may be crosslinked intramolecularly or intermolecularly among the extracellular matrix molecules that make up the extracellular matrix component. When the extracellular matrix component includes a fragmented extracellular matrix component, at least a portion of the fragmented extracellular matrix component may be crosslinked intermolecularly or intramolecularly.

[0061] An extracellular matrix component at least partially intermolecularly or intramolecularly crosslinked can be produced, for example, by a method including a step of crosslinking an extracellular matrix component (a crosslinking step). The extracellular matrix component can include, for example, a fragmented and crosslinked extracellular matrix component. The fragmented and crosslinked extracellular matrix component can be produced, for example, by a method including, in this order, a step of fragmenting an extracellular matrix component and a step of crosslinking the fragmented extracellular matrix component, or a method including, in this order, a step of crosslinking an extracellular matrix component and a step of fragmenting the crosslinked extracellular matrix component.

[0062] Examples of the crosslinking method include physical crosslinking by applying heat, ultraviolet light, radiation, etc., and chemical crosslinking by a crosslinking agent, enzyme reaction, etc., but the method is not particularly limited. From the viewpoint of not interfering with cell growth, physical crosslinking is preferred. Crosslinking (physical crosslinking and chemical crosslinking) may be crosslinking via a covalent bond.

[0063] When the extracellular matrix component includes a collagen component, crosslinks may be formed between collagen molecules (triple helix structure) or between collagen fibrils formed by collagen molecules. Crosslinks may be thermal crosslinks (thermal crosslinks). Thermal crosslinks can be performed, for example, by heating under reduced pressure using a vacuum pump. When thermal crosslinking of collagen components is performed, the extracellular matrix component may be crosslinked by forming a peptide bond (-NH-CO-) between an amino group of a collagen molecule and a carboxy group of the same or another collagen molecule.

[0064] The extracellular matrix components can also be crosslinked by using a crosslinking agent. The crosslinking agent may be, for example, one capable of crosslinking a carboxyl group with an amino group, or one capable of crosslinking amino groups with each other. As the crosslinking agent, for example, aldehyde-based, carbodiimide-based, epoxide-based, and imidazole-based crosslinking agents are preferable from the viewpoints of economy, safety, and operability. Specific examples of the crosslinking agent include water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide sulfonate.

[0065] The degree of crosslinking can be determined appropriately depending on the type of extracellular matrix component, 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. When the degree of crosslinking is within the above range, the extracellular matrix molecules can be appropriately dispersed, and the redispersibility after dry storage is good.

[0066] When amino groups in the extracellular matrix components are used for crosslinking, the degree of crosslinking can be quantified based on the TNBS (2,4,6-trinitrobenzenesulfonic acid) method described in Acta Biomaterialia, 2015, vol. 25, pp. 131-142, etc. The degree of crosslinking by the TNBS method may be within the above-mentioned range. The degree of crosslinking by the TNBS method is the ratio of amino groups used for crosslinking among the amino groups in the extracellular matrix. When the extracellular matrix components include collagen components, it is preferable that the degree of crosslinking measured by the TNBS method is within the above-mentioned range.

[0067] The degree of crosslinking may be calculated by quantifying the carboxyl groups. For example, in the case of a water-insoluble extracellular matrix component, the degree of crosslinking may be quantified by the TBO (toluidine blue O) method. The degree of crosslinking by the TBO method may be within the above-mentioned range.

[0068] In the crosslinking step, the temperature (heating temperature) and time (heating time) when heating the extracellular matrix component can be appropriately determined. 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, or 220°C. The heating time (the time to hold at the above heating temperature) can be appropriately set 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 step, heating may be performed in the absence of a solvent, or under reduced pressure conditions.

[0069] The content of extracellular matrix components in the three-dimensional tissue structure may be, based on the dry weight of the three-dimensional tissue structure, 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, 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 content of the extracellular matrix components in the three-dimensional tissue structure may be 0.01 to 90 mass%, 10 to 90 mass%, 10 to 80 mass%, 10 to 70 mass%, 10 to 60 mass%, 1 to 50 mass%, 10 to 50 mass%, 10 to 30 mass%, or 20 to 30 mass%, based on the dry weight of the three-dimensional tissue structure.

[0070] The collagen component in the three-dimensional tissue structure may be defined by its area ratio or volume ratio. "Defining by area ratio or volume ratio" means, for example, making the collagen component in the three-dimensional tissue structure distinguishable from other tissue components by a known staining method (e.g., immunostaining using an anti-collagen antibody or Masson's trichrome staining), and then calculating the ratio of the area in which the collagen component is present in the entire three-dimensional tissue structure using macroscopic observation, various microscopes, image analysis software, etc. When defining by area ratio, there is no limitation on which cross section or surface in the three-dimensional tissue structure defines the area ratio, but for example, when the three-dimensional tissue structure is a sphere, it may be defined by a cross section passing through the approximate center of the sphere.

[0071] For example, when the collagen component in the three-dimensional tissue structure is defined by the area ratio, the area ratio is usually 0.01 to 99% based on the total area of ​​the three-dimensional tissue structure, and may be 1 to 99%, 5 to 90%, 7 to 90%, 20 to 90%, 30 to 90%, or 50 to 90%. The area ratio of the collagen component can be calculated, for example, by staining the obtained three-dimensional tissue structure with Masson's trichrome and calculating the ratio of the area of ​​the collagen component stained blue to the total area of ​​a cross section passing through approximately the center of the three-dimensional tissue structure.

[0072] The three-dimensional tissue structure may contain a polymer electrolyte. The polymer electrolyte is a polymer compound having electrolyte properties. Examples of the polymer electrolyte include glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid, or derivatives thereof, but are not limited thereto. The polymer electrolyte may be one of the above-mentioned substances, or may contain two or more of them in combination.

[0073] The polyelectrolyte is preferably a glycosaminoglycan, more preferably contains at least one selected from the group consisting of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate, and even more preferably contains heparin. When the three-dimensional tissue structure contains a polyelectrolyte, excessive aggregation of extracellular matrix components can be more effectively suppressed, and as a result, the desired three-dimensional tissue structure is more easily obtained. When the three-dimensional tissue structure contains heparin, the effect becomes even more remarkable.

[0074] The ratio (C2 / C1) of the mass C2 of the polymer electrolyte to the mass C1 of the extracellular matrix component may be 1 / 100 to 100 / 1, 1 / 10 to 10 / 1, 1 / 5 to 5 / 1, or 1 / 2 to 2 / 1, or may be 1 / 1.5 to 1.5 / 1.

[0075] The thickness of the three-dimensional tissue structure according to the present embodiment may be 10 μm or more, 30 μm or more, 50 μm or more, 100 μm or more, 300 μm or more, 600 μm or more, or 1000 μm or more. Such a three-dimensional tissue structure has a structure closer to living tissue, and is suitable as a substitute for laboratory animals and as a transplant material. The upper limit of the thickness of the three-dimensional tissue structure is not particularly limited, and may be, for example, 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0076] Here, the "thickness of the three-dimensional tissue structure" means the distance between both ends in the direction perpendicular to the main surface when the three-dimensional tissue structure is a rectangular parallelepiped. When the main surface has projections and recesses, the thickness means the distance at the thinnest part of the main surface.

[0077] When the three-dimensional tissue structure is spherical or approximately spherical, the thickness of the three-dimensional tissue structure means the diameter of the three-dimensional tissue structure. When the three-dimensional tissue structure is ellipsoidal or approximately ellipsoidal, the thickness of the three-dimensional tissue structure means the minor axis of the three-dimensional tissue structure. When the three-dimensional tissue structure is approximately spherical or approximately ellipsoidal and has an uneven surface, the thickness of the three-dimensional tissue structure means the shortest distance between two points where a straight line passing through the center of gravity of the three-dimensional tissue structure intersects with the surface.

[0078] The three-dimensional tissue structure according to this embodiment is constructed in a cell culture vessel. The cell culture vessel is not particularly limited as long as it is capable of constructing a three-dimensional tissue structure and culturing the constructed three-dimensional tissue structure. Specific examples of the cell culture vessel include dishes, cell culture inserts (e.g., Transwell (registered trademark) inserts, Netwell (registered trademark) inserts, Falcon (registered trademark) cell culture inserts, Millicell (registered trademark) cell culture inserts, etc.), tubes, flasks, bottles, plates, etc. In constructing a three-dimensional tissue structure, dishes or various cell culture inserts are preferred from the viewpoint of enabling more appropriate evaluation using the three-dimensional tissue structure.

[0079] [Method for producing a three-dimensional structure] The method for producing a three-dimensional tissue structure according to one embodiment includes a step of mixing cells and a matrix precursor containing biocompatible molecules (hereinafter, also simply referred to as "matrix precursor") in an aqueous medium, and then forming a matrix from the matrix precursor (forming step A). ​​The cells are as described above.

[0080] The matrix precursor containing a biocompatible molecule is a substance capable of forming a matrix containing a biocompatible molecule, and refers to a substance in a state in which the matrix is ​​not formed. Examples of the matrix containing a biocompatible molecule include those mentioned above, and examples of the matrix precursor include the above biocompatible molecules in a state in which the matrix is ​​not formed.

[0081] The matrix precursor concentration in the aqueous medium may be 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, 0.5 mg / mL or more, 0.6 mg / mL or more, 0.7 mg / mL or more, 0.8 mg / mL or more, 0.9 mg / mL or more, or 1.0 mg / mL or more, based on the total amount of the aqueous medium. The matrix precursor concentration in the aqueous medium may be 10.0 mg / mL or less, 8.0 mg / mL or less, 6.0 mg / mL or less, 5.0 mg / mL or less, 3.0 mg / mL or less, 1.0 mg / mL or less, 0.8 mg / mL or less, or 0.6 mg / mL or less, based on the total amount of the aqueous medium.

[0082] When the matrix precursor comprises two or more substances, for example a combination of fibrinogen and thrombin, the aqueous medium comprising cells and matrix precursor may be obtained, for example, by mixing a first aqueous medium comprising cells and a first matrix precursor (e.g., thrombin) with a second aqueous medium comprising a second matrix precursor (e.g., fibrinogen). The second aqueous medium may comprise cells, or both the first and second aqueous media may comprise cells.

[0083] In the formation step A, when a matrix containing an inhibitor of degradation by a substance secreted by cells is formed, the matrix may be formed after mixing the cells, the matrix precursor, and the inhibitor. The inhibitor of degradation by a substance secreted by cells is as described above.

[0084] The term "aqueous medium" means a liquid containing water as an essential component. The aqueous medium may be, for example, an aqueous medium containing a cationic substance. The aqueous medium containing a cationic substance may be, for example, a cationic buffer solution such as Tris-hydrochloric acid buffer, Tris-maleic acid buffer, Bis-Tris buffer, or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or may be a medium containing water and a cationic compound such as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, or polyarginine as a cationic substance. In addition, a culture medium may be used as the aqueous medium. Examples of the culture medium include liquid culture media such as Dulbecco's Modified Eagle medium (DMEM). The liquid culture medium may be a mixed culture medium in which two types of culture media are mixed.

[0085] The concentration and pH of the cationic substance (e.g., Tris in Tris-HCl buffer) in the aqueous medium containing the cationic substance are not particularly limited as long as they do not adversely affect cell growth and the construction of a three-dimensional tissue structure. For example, the concentration of the cationic substance may be 10 to 100 mM, or 40 to 70 mM, or 50 mM based on the total amount of the aqueous medium containing the cationic substance. The pH of the aqueous medium (e.g., cationic buffer) may be 6.0 to 8.0, 6.8 to 7.8, or 7.2 to 7.6.

[0086] For example, when the matrix is ​​in the form of a hydrogel, hydrogel fiber, or hydrogel film, "forming a matrix" may mean causing gelation.

[0087] Formation of the matrix may involve incubation for a period of time.

[0088] The cell density in the aqueous medium in the formation step A can be appropriately determined depending on the shape, thickness, etc. of the intended three-dimensional tissue structure. For example, the cell density in the medium in the formation step A is 1 to 10 8 cells / mL, 3 ~10 7 It may be cells / mL.

[0089] The formation of the matrix may be carried out after further mixing with an extracellular matrix component, which is as described above.

[0090] In one embodiment of the method for producing a three-dimensional tissue structure, when forming a matrix, cells may be suspended in advance once in an aqueous medium containing an extracellular matrix component and a cationic substance, and after removing the aqueous medium, the cells may be resuspended in an aqueous medium containing a matrix precursor to form a matrix. When this step is performed, cells tend to accumulate with each other inside the matrix.

[0091] The formation of the matrix may be carried out after further mixing a polymer electrolyte. As the polymer electrolyte, the above-mentioned ones can be used.

[0092] In one embodiment of the method for producing a three-dimensional tissue structure, when forming a matrix, cells may be suspended in advance in an aqueous medium containing an extracellular matrix component, a polyelectrolyte, and a cationic substance, and after removing the aqueous medium, the cells may be resuspended in an aqueous medium containing a matrix precursor to form a matrix. When this step is performed, cells tend to accumulate with each other inside the matrix.

[0093] The method for producing a three-dimensional tissue structure according to this embodiment may further include a step of culturing the cells in the formed matrix (culture step). By further including the culture step, a three-dimensional tissue structure containing cells and the matrix can be produced. The culture can be carried out, for example, by adding a medium to the matrix containing the cells.

[0094] The medium used in the culture step can be appropriately selected depending on the type of cells, and examples thereof include Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RPMI, GlutaMax medium, and medium specifically for vascular endothelial cells (EGM2). The medium may be a medium supplemented with serum or a serum-free medium. The medium may be a medium supplemented with growth factors. The medium may be a mixed medium in which two types of media are mixed.

[0095] The culture temperature in the culture step may be, for example, 20° C. to 40° C., or 30° C. to 37° C. The pH of the medium may be 6 to 8, or 7.2 to 7.4. The culture time may be 1 day to 2 weeks, or 1 week to 2 weeks.

[0096] The culture vessel (support) is not particularly limited, and may be, for example, a dish, a well insert, a low-adhesion plate, or a plate having a bottom shape such as U-shaped or V-shaped. The cells may be cultured while attached to the support, or may be cultured without being attached to the support, or may be cultured after being detached from the support during the culture. When the cells are cultured without being attached to the support, or when the cells are cultured after being detached from the support during the culture, it is preferable to use a plate having a bottom shape such as U-shaped or V-shaped that inhibits adhesion of the cells to the support, or a low-adhesion plate.

[0097] The cell density in the medium in the culture step can be appropriately determined depending on the shape and thickness of the intended three-dimensional tissue structure, the size of the culture vessel, etc. For example, the cell density in the medium in the culture step can be set to 1 to 10 8 cells / mL, 3 ~10 7 The cell density in the medium in the culturing step may be the same as the cell density in the aqueous medium in the formation step.

[0098] A method for producing a three-dimensional tissue structure according to one embodiment includes a step of preparing a plurality of tissue bodies containing cells (preparation step), and a step of forming a matrix between the plurality of tissue bodies from a matrix precursor containing biocompatible molecules (formation step B). The matrix is ​​as described above. By this method, a three-dimensional tissue structure such as the three-dimensional tissue structure 100 in FIG. 1 can be produced.

[0099] The preparation step is a step of forming and preparing a plurality of tissue structures, and the above-mentioned second part can be produced by the preparation step. In the preparation step, the method of forming the tissue structures is not particularly limited, and can be performed by a method well known to those skilled in the art. The preparation step may include culturing cells in a medium. The culture of cells in the medium can be performed in the same manner as the above-mentioned culture step.

[0100] The preparing step may include contacting the cells with an extracellular matrix component and / or a polyelectrolyte prior to culturing the cells, in which case the culturing comprises culturing the cells in contact with the extracellular matrix component and / or the polyelectrolyte.

[0101] The preparation step may include embedding the cells in a hydrogel after contacting the cells with the extracellular matrix component and / or the polymer electrolyte and before culturing the cells. Embedding in a hydrogel can be performed, for example, by preparing a composition containing the cells and a pre-gelled hydrogel and gelling the composition. Gelation of the composition containing the cells and the hydrogel-forming substance may be performed in the above-mentioned aqueous medium. The hydrogel does not have resistance to degradation by a substance secreted by the cells in the tissue. By embedding the cells in the hydrogel, a tissue having a three-dimensional structure can be formed.

[0102] When cells are embedded in a hydrogel, the composition containing the cells and the pre-gelled hydrogel may contain the above-mentioned extracellular matrix components and / or polyelectrolytes.

[0103] In the formation step B, a matrix is ​​formed between the multiple tissues prepared in the preparation step. By performing the formation step B, the multiple tissue bodies prepared in the preparation step can be connected and assembled, and a three-dimensional tissue structure in which the second portions and the first portions are alternately in contact with each other can be produced.

[0104] The formation step B can be carried out, for example, by forming a matrix from a matrix precursor in a state where a plurality of tissues are in contact with each other via the matrix precursor. Alternatively, the formation step B may be carried out by forming a matrix in contact with one tissue, and then forming the matrix from the matrix precursor in a state where the matrix and the tissues are in contact with each other via the matrix precursor. When the form of the matrix is ​​a hydrogel and / or a hydrogen fiber, "forming a matrix" may mean gelling.

[0105] The formation of the matrix in the formation step B can be carried out in the same manner as in the formation step A, for example, by preparing an aqueous medium containing a matrix precursor and forming a matrix from the matrix precursor in the aqueous medium.

[0106] [Unit for forming three-dimensional tissue structure] The above-mentioned matrix can be suitably used for forming a three-dimensional tissue structure according to the present invention. Thus, as one embodiment of the present invention, there is provided a unit for forming a three-dimensional tissue structure, which comprises a matrix containing a biocompatible molecule, and the matrix is ​​resistant to degradation by substances secreted by cells.

[0107] The unit for forming a three-dimensional tissue structure according to one embodiment can be used to form a three-dimensional tissue structure, for example, by assembling the unit and tissue bodies containing cells while alternately connecting them to each other. Therefore, the unit for forming a three-dimensional tissue structure can also be regarded as a support material for forming a three-dimensional tissue structure. EXAMPLES

[0108] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these.

[0109] The cells, reagents and preparation methods used in the production of the three-dimensional tissue structure are shown in Tables 1 to 6 below.

[0110] [Table 1]

[0111] [Table 2]

[0112] [Table 3]

[0113] [Table 4]

[0114] [Table 5]

[0115] [Table 6]

[0116] [Test Example 1: Comparison of long-term culture performance in three-dimensional tissue constructs] <Comparative Example 1: Production of a three-dimensional tissue structure by three-dimensional culture> The cells that make up tissue 1 are NHDF and HUVEC, each of which is 1.2 x 10 7 The cells were suspended in a mixed solution (mixing ratio 1:1 (v / v)) of 50 mM Tris-HCl buffer (pH 7.4) solution in which 0.05 mg / mL heparin was dissolved, and 50 mM Tris-HCl buffer (pH 7.4) solution in which collagen was dissolved. The suspension was centrifuged at room temperature at 1000×g for 1 minute, and the supernatant was removed. Next, a general-purpose medium was added to precipitate the cells, and the cells were suspended. The resulting suspension was then added to a Transwell culture insert and cultured to obtain a three-dimensional tissue structure by three-dimensional culture. The cell recovery rate was calculated from the following formula on days 7, 14, 21, and 28 after the start of cell culture. During the culture period, the medium was replaced twice a week (once every 3 days and once every 4 days). Cell recovery rate (%) = number of live cells on the day of measurement / number of cells added x 100

[0117] <Example 1: Production of a three-dimensional tissue structure according to one embodiment of the present invention> (1. Manufacturing of tissues) The cells that make up tissue 1 are NHDF and HUVEC, each of which is 1.2 x 10 7The cells were suspended in a mixture of 50 mM Tris-HCl buffer (pH 7.4) solution in which 0.05 mg / mL heparin was dissolved, and 50 mM Tris-HCl buffer (pH 7.4) solution in which collagen was dissolved (mixing ratio 1:1 (v / v)). The suspension was centrifuged at room temperature at 1000×g for 1 minute, and the supernatant was removed. Next, a general-purpose medium was added to precipitate the cells, and the cells were suspended. The obtained suspension was mixed with a 10 mg / mL fibrinogen solution prepared using DMEM in a ratio of 1:1 (v / v) to obtain a mixture. The obtained mixture was mixed with a 10 Unit / mL thrombin solution prepared using DMEM in a ratio of 2:1 (v / v) to obtain a cell suspension. The first container was placed on the second container, and 150 μL of the cell suspension was seeded in the first container. The cell suspension was left to stand in a CO2 incubator (37° C., 5% CO2) until it gelled. After adding an appropriate amount of the dedicated medium to the inside of the first container and the second container, the mixture was cultured in a CO2 incubator (37°C, 5% CO2) for 3 days to obtain tissue construct 1. A total of six tissue constructs 1 were obtained in the same manner.

[0118] (2. Production of a three-dimensional tissue structure according to one embodiment of the present invention) A three-dimensional tissue structure was produced by bonding six tissue bodies 1 in a horizontal x vertical: 2 x 3 arrangement via a matrix (a hydrogel made by gelling a mixed solution of thrombin / fibrinogen / α2-antiplasmin) using the following procedure. (i) One tissue structure 1 (first tissue structure 1) was removed from the first container, and 20 μL of a mixed solution of 3.3 Units / mL thrombin, 20 mg / mL fibrinogen, and 100 μg / mL α2-antiplasmin (hereinafter also referred to as "thrombin / fibrinogen / α2-antiplasmin mixed solution") was added to the surface to be laminated, and the mixture was left to stand until it gelled, thereby obtaining a tissue structure 1 in which the surface to be laminated was covered with gel. (ii) After gelation, 20 μL of a thrombin / fibrinogen / α2-antiplasmin mixed solution was added to the surface to which another tissue structure 1 was to be attached (all or a part of the surface covered with the gel). (iii) Next, another tissue body 1 (second tissue body 1) was removed from the first container, and the tissue body 1 (second tissue body 1) removed in (ii) was placed on the surface to which the thrombin / fibrinogen / α2-antiplasmin mixed solution had been added and left to stand until it gelled, thereby obtaining a three-dimensional tissue structure in which the first tissue body 1 and the second tissue body 1 were adhered together via a matrix (hydrogel formed from the gelled thrombin / fibrinogen / α2-antiplasmin mixed solution). (iv) Next, 20 μL of thrombin / fibrinogen / α2-antiplasmin mixed solution was added to the surface of the second tissue body 1 to be laminated in the three-dimensional tissue structure obtained in (iii) (part or all of the surface other than the surface adhered to the first tissue body 1) and left to stand until it gelled, resulting in the surface of the second tissue body 1 to be laminated being covered with gel. (v) The above steps (ii) to (iv) were repeated to obtain a three-dimensional tissue structure in which six tissue bodies 1 were bonded together in a horizontal×vertical: 2×3 arrangement via the matrix. The obtained three-dimensional tissue structure of Example 1 was then cultured in a general-purpose medium for 25 days, and the cell recovery rate was calculated in the same manner as in Comparative Example 1 on days 7, 14, 21, and 28 (including the three-day culture period required for producing the tissue structure) from the start of cell culture.

[0119] <Example 2: Production of a three-dimensional tissue structure according to one embodiment of the present invention> (1. Preparation of fibrin fibers) 100 mL of a solution containing 3.3 Unit / mL thrombin and 20 mg / mL fibrinogen was gelled to obtain a fibrin gel. 20 mL of hexafluoroisopropanol was added to the obtained fibrin gel to obtain a fibrin solution. Next, the obtained fibrin solution was fiberized using an electrospinning device under the following conditions to produce a nonwoven fabric of fibrin fibers. Metal nozzle: Stainless steel nozzle with inner diameter of 0.2 mm and length of 1 cm DC current: 20kV Spout rate: 4.4mL / min

[0120] (2. Manufacturing of Organisms) A total of six tissue structures 1 were obtained in the same manner as in Example 1 (1. Production of tissue structure).

[0121] (3. Production of a three-dimensional tissue structure according to one embodiment of the present invention) The following procedure was used to produce a three-dimensional tissue structure in which six tissue bodies 1 were adhered together in a horizontal x vertical: 2 x 3 matrix (containing a hydrogel of a gelled mixture of thrombin / fibrinogen / α2-antiplasmin within the nonwoven fabric described above). (i) One tissue body 1 (first tissue body 1) is taken out from the first container. (ii) 20 μL of a thrombin / fibrinogen / α2-antiplasmin mixed solution is added to the surface of the first tissue body 1 to be laminated. (iii) A nonwoven fabric of fibrin fibers was placed on the surface to which the other tissue body 1 was to be attached (all or part of the surface to which the thrombin / fibrinogen / α2-antiplasmin mixed solution had been added) and left to stand until the thrombin / fibrinogen / α2-antiplasmin mixed solution gelled. (iv) 20 μL of the thrombin / fibrinogen / α2-antiplasmin mixed solution is added to the nonwoven fabric placed in (iii). (v) Next, another tissue body 1 (second tissue body 1) was removed from the first container, and the tissue body 1 (second tissue body 1) removed in (iv) was placed on the nonwoven fabric to which the thrombin / fibrinogen / α2-antiplasmin mixed solution had been added and left to stand until it gelled, thereby obtaining a three-dimensional tissue structure in which the first tissue body 1 and the second tissue body 1 were adhered together via a matrix (a hydrogel formed by gelling the thrombin / fibrinogen / α2-antiplasmin mixed solution within the nonwoven fabric). (vi) The above steps (ii) to (v) were repeated to obtain a three-dimensional tissue structure in which six tissue bodies 1 were bonded in a horizontal×vertical: 2×3 configuration via the matrix. The obtained three-dimensional tissue structure of Example 2 was cultured in a general-purpose medium for 25 days, and the cell recovery rate was calculated in the same manner as in Comparative Example 1 on days 7, 14, 21, and 28 (including the three-day culture period required for producing the tissue structure) from the start of cell culture.

[0122] <Comparison of long-term culture performance of three-dimensional tissue constructs> The calculated cell recovery rates were compared for the three-dimensional tissue structures of Comparative Example 1 and Examples 1 and 2. The results are shown in Table 7. A cell recovery rate of 70% or more was judged to be able to maintain culture and was rated as "O", whereas a cell recovery rate of less than 70% was judged to be unable to maintain culture and was rated as "X".

[0123] [Table 7]

[0124] As shown in Table 7, the three-dimensional tissue structure of Comparative Example 1 had a cell recovery rate of less than 70% (23.1%) on day 7, and was unable to maintain cell proliferation. In contrast, the three-dimensional tissue structures of Examples 1 and 2 had cell recovery rates of 70% or more up to day 28, and were able to maintain cell proliferation.

[0125] [Test Example 2: Comparison of functional maintenance performance of three-dimensional tissue constructs] <Comparative Example 2: Production of a three-dimensional tissue structure by three-dimensional culture> The cell types constituting tissue bodies 1, 2, and 3, namely NHDF, HUVEC, BFP-HUVEC, GFP-BPDC, and RFP-BCAF, were each 4 × 10 6 cells, 8 x 10 6 cells, 8 x 10 6 cells and 4 x 10 6 Except for using cells, a three-dimensional tissue structure of Comparative Example 2 was obtained in the same manner as in Comparative Example 1. On the seventh day after the start of cell culture, evaluation of cancer cell proliferation, gene expression analysis of CAF and CAE marker proteins, which are cancer stromal cell markers, and evaluation of vascular network formation were performed.

[0126] Cancer cell proliferation was evaluated as follows: After dispersing the three-dimensional tissue structure with trypsin, the number of fluorescent positive cells was counted as the number of cancer cells using a fluorescent cell counter CountessIIFL. Then, the number of cancer cells after culture / the number of cancer cells input was calculated.

[0127] Gene expression analysis was performed as follows. 700 μL of QIAzol Lysis Reagent (QIAGEN) was added to the three-dimensional tissue structure, and the cells were disrupted by centrifugation after being subjected to a QIAshredder column (QIAGEN). Then, 20% (vol) of chloroform (Fuji film) was added to the filtrate, mixed thoroughly, and centrifuged. After separating the supernatant, 1.5 times the amount of 100% ethanol (Fuji film) was added to precipitate total RNA, and RNA was purified using miRNeasy Micro Kit (Qiaegn). Finally, 20 μL of nuclease free water was added to elute the total RNA. Microarray gene expression analysis was performed using SurePrint G3 Human GE Microarray 8 × 60K Ver.3.0 (Agilent). Using 100 ng of purified total RNA as a template, labeling and hybridization were performed by the one-color method using the One-Color RNA Spike-In Kit, Low Input Quick Amp Labeling Kit (1color) and Expression Hybridization Kit (Agilent), and scanning was performed with a DNA Microarray Scanner (Agilent). For expression data, the acquired image data was digitized using Feature Extraction (Agilent) software, and then normalized between arrays using GeneSpring GX 14.9.1 (Agilent) software.

[0128] The formation of vascular networks was evaluated as follows: Fluorescent proteins expressed in vascular endothelial cells were observed using a confocal microscope system (PerkinElmer Operetta CLS) in live cell fluorescence mode (37°C, 5% CO2) to evaluate the presence or absence of network structure formation and its maintenance during the culture period (culture days 4, 7, 14, 21, and 28).

[0129] <Example 3: Production of a three-dimensional tissue structure according to one embodiment of the present invention> In the same manner as in Example 1, two tissue bodies each of tissue bodies 1 to 3 were produced in total, i.e., six tissue bodies were bonded in a horizontal x vertical: 2 x 3 arrangement via a matrix (a hydrogel formed by gelling a mixed solution of thrombin / fibrinogen / α2-antiplasmin), to produce a three-dimensional tissue structure. In the obtained three-dimensional tissue structure, the same tissue bodies were arranged horizontally, and tissue body 1, tissue body 2, and tissue body 3 were arranged vertically in this order. Next, the obtained three-dimensional tissue structure of Example 3 was cultured in a general-purpose medium for four days, and on the seventh day after the start of cell culture (including the three-day culture period required for the production of the tissue bodies), the evaluation of cancer cell proliferation, gene expression analysis of CAF and CAE marker proteins, which are cancer stromal cell markers, and evaluation of vascular network formation were performed in the same manner as in Comparative Example 2.

[0130] Example 4: Production of a three-dimensional tissue structure according to one embodiment of the present invention A total of six tissue bodies, two each of tissue bodies 1 to 3, were produced in the same manner as in Example 2. Furthermore, a three-dimensional tissue structure was produced in which the six tissue bodies were bonded in a horizontal x vertical: 2 x 3 arrangement via a matrix (containing a hydrogel in which a thrombin / fibrinogen / α2-antiplasmin mixed solution was gelled within the nonwoven fabric) in the same manner as in Example 2. The obtained three-dimensional tissue structure had identical tissue bodies lined up in the horizontal direction, and tissue body 1, tissue body 2, and tissue body 3 lined up in that order in the vertical direction. Next, the obtained three-dimensional tissue structure of Example 4 was cultured in a general-purpose medium for four days, and on the seventh day after the start of cell culture (including the three-day culture period required for producing the tissue), evaluation of cancer cell proliferation, gene expression analysis of CAF and CAE marker proteins, which are cancer stromal cell markers, and evaluation of vascular network formation were performed in the same manner as in Comparative Example 2.

[0131] <Comparison of functional maintenance performance of three-dimensional tissue constructs> Regarding cancer cell proliferation, if the number of cancer cells after culture / the number of cancer cells input was 1.2 or more, it was judged that proliferation was possible and was evaluated as "〇", and if it was less than 1.2, it was judged that proliferation was impossible and was evaluated as "×". Regarding the maintenance of CAF and CAE marker proteins, if the gene expression level after culture / gene expression level before culture was 0.8 or more for all items in the gene expression analysis results of CAF marker protein expression genes (2 items, ACTA2 and FAP genes) and CAE marker protein expression genes (2 items, ANPEP and ITGAV genes), it was judged that maintenance was possible and was evaluated as "〇", and if it was less than 0.8, it was judged that maintenance was impossible and was evaluated as "×". Regarding vascular network formation, if the structure formation and maintenance during the culture period were confirmed, it was evaluated as "〇", and if the structure formation and maintenance during the culture period were not confirmed, it was evaluated as "×". The results are shown in Table 8.

[0132] [Table 8]

[0133] As shown in Table 8, the three-dimensional tissue structure of Comparative Example 2 was capable of supporting the proliferation of cancer cells, but the expression of CAF and CAE markers was not maintained, indicating that the proliferation of cancer stromal cells could not be maintained. Furthermore, the formation of a vascular network structure and its maintenance during the culture period could not be confirmed, and it was confirmed that the formation of a vascular network was inhibited in the co-culture system. In contrast, the three-dimensional tissue structures of Examples 3 and 4 were evaluated as excellent in all items, indicating that the inhibition of the formation of functional structures and the inhibition of cell proliferation were reduced.

[0134] [Test Example 3: Comparison of thickness maintenance performance by long-term culture in three-dimensional tissue constructs] <Comparative Example 3: Production of a three-dimensional tissue structure by three-dimensional culture> In the same manner as in Comparative Example 2, a three-dimensional structure of Comparative Example 3 was obtained.

[0135] <Example 5: Production of a three-dimensional tissue structure according to one embodiment of the present invention> A total of six tissue bodies, two each of tissue bodies 1 to 3, were produced in the same manner as in Example 4. Furthermore, a three-dimensional tissue structure was produced in which the six tissue bodies were bonded in a horizontal x vertical: 2 x 3 arrangement via a matrix (containing a hydrogel in which a thrombin / fibrinogen / α2-antiplasmin mixed solution was gelled within the nonwoven fabric) in the same manner as in Example 4. The obtained three-dimensional tissue structure had identical tissue bodies lined up in the horizontal direction, and tissue body 1, tissue body 2, and tissue body 3 lined up in that order in the vertical direction.

[0136] <Comparison of thickness maintenance performance of three-dimensional tissue constructs> The obtained three-dimensional tissue structures of Comparative Example 3 and Example 5 were cultured in a general-purpose medium for 21 days, and on the 7th and 21st days after the start of cell culture (including the 3-day culture period required for the production of the tissue in the case of the three-dimensional tissue structure of Example 5), each three-dimensional tissue structure was fixed using a formalin buffer solution (model number "062-01661", Fujifilm Wako Pure Chemical Industries, Ltd.). Next, each three-dimensional tissue structure was removed from the cell culture insert and embedded in paraffin, and then thin sections were prepared along a line passing through the center of gravity when viewed from the top of the three-dimensional tissue structure (top of the cell culture insert). Next, the thin sections were stained with hematoxylin-eosin (HE) and observed under a microscope, and the maximum thickness of the three-dimensional tissue structure was measured. This allowed evaluation of thickness maintenance during long-term culture.

[0137] Regarding thickness maintenance through long-term culture, if the ratio of thickness of the three-dimensional tissue structure on the 21st day of culture to the thickness of the three-dimensional tissue structure on the 7th day of culture was 0.8 or more, it was judged that the thickness could be maintained and was evaluated as "O", and if it was less than 0.8, it was judged that the thickness could not be maintained and was evaluated as "X".

[0138] [Table 9]

[0139] As shown in Table 9, the thickness was not maintained in the three-dimensional tissue structure of Comparative Example 3. In contrast, the thickness of the three-dimensional tissue structure of Example 5 was maintained for a long period of time.

[0140] 100...three-dimensional tissue structure, 10...first part, 20...second part, 30...luminal structure.

Claims

1. a matrix containing cells and biocompatible molecules, The matrix is ​​resistant to degradation by substances secreted by the cells.

2. The three-dimensional tissue structure according to claim 1 , wherein the matrix contains an inhibitor of degradation by a substance secreted by the cells.

3. The three-dimensional tissue structure according to claim 1 , wherein the biocompatible molecule is resistant to degradation by substances secreted by the cells.

4. The method includes the steps of: (a) providing a first portion or portions each of which is made of the matrix; and (b) providing a second portion or portions each of which is made of a tissue structure containing the cells; The three-dimensional tissue structure according to claim 1 , wherein each of the second portions is disposed so as to be in contact with another second portion via the first portion.

5. The three-dimensional tissue structure according to claim 1 , wherein the matrix allows humoral factors to pass through but does not allow the cells to pass through.

6. The three-dimensional tissue structure according to claim 1 , wherein the matrix is ​​in the form of one or more selected from the group consisting of a hydrogel, a hydrogel fiber, and a hydrogel film.

7. The three-dimensional structure according to claim 1, which has a thickness of 600 μm or more.

8. The method includes the steps of mixing cells with a matrix precursor containing a biocompatible molecule in an aqueous medium, and then forming a matrix from the matrix precursor, The method for producing a three-dimensional tissue structure, wherein the matrix is ​​resistant to degradation by substances secreted by the cells.

9. The method of claim 8 , further comprising culturing the cells in the formed matrix.

10. The method includes the steps of: preparing a plurality of tissue structures containing cells; and forming a matrix between the plurality of tissue structures from a matrix precursor containing a biocompatible molecule; The method for producing a three-dimensional tissue structure, wherein the matrix is ​​resistant to degradation by substances secreted by the cells.

11. The method for producing a three-dimensional tissue structure according to claim 8 or 10, wherein the matrix contains an inhibitor of degradation by a substance secreted by the cells.

12. The method for producing a three-dimensional tissue structure according to claim 8 or 10, wherein the biocompatible molecule is resistant to degradation by a substance secreted by the cells.

13. The method for producing a three-dimensional tissue structure according to claim 8 or 10, wherein the matrix is ​​in the form of one or more selected from the group consisting of a hydrogel, a hydrogel fiber, and a hydrogel film.

14. a matrix containing biocompatible molecules; The matrix is ​​resistant to degradation by substances secreted by cells, in the unit for forming a three-dimensional tissue structure.

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