Three-dimensional cell tissue production method and three-dimensional cell tissue

Abstract

This three-dimensional cell tissue production method comprises: a step (a) for obtaining a stromal cell-containing mixture containing stromal cells, a cationic substance, an extracellular matrix component, and a polymer electrolyte; a step (b) for gelling the stromal cell-containing mixture to obtain a gel-like composition; a step (c) for obtaining a target-cell-containing mixture containing target cells, a cationic substance, an extracellular matrix component, and a polymer electrolyte; a step (d) for placing the target-cell-containing mixture so as to be in contact with the gel-like composition containing stromal cells; a step (e) for gelling the target-cell-containing mixture to obtain a gel-like composition; and a step (f) for incubating the gel-like composition containing the stromal cells and the gel-like composition containing the target cells to obtain three-dimensional cell tissue. In the three-dimensional cell tissue, the total volume of the target cells accounts for 60 vol% or more of the volume of the gel-like composition containing the target cells.

Description

Method for manufacturing three-dimensional cell tissue and three-dimensional cell tissue

[0001] The present invention relates to a method for producing a three-dimensional cellular tissue and a three-dimensional cellular tissue. This application claims priority to Japanese Patent Application No. 2023-122401, filed on July 27, 2023, the contents of which are incorporated herein by reference.

[0002] In recent years, in fields such as regenerative medicine and drug assay systems that require an environment similar to that of a living body, the use of three-dimensional cellular tissues organized in three dimensions has been shown to be superior to cells grown on flat plates. For this reason, various techniques have been developed for constructing three-dimensional cellular tissues in vitro.

[0003] The present inventors have previously developed a technology for producing three-dimensional cellular tissues (see, for example, Patent Document 1), which includes the steps of obtaining a mixture in which cells are suspended in a solution containing at least a cationic buffer, an extracellular matrix component, and a polyelectrolyte, collecting the cells from the obtained mixture to form a cell aggregate on a substrate, and culturing the cells to obtain a three-dimensional cellular tissue. The three-dimensional cellular tissues can be used, for example, as biological tissue models, solid cancer models, etc., and can be used in various assays such as drug screening.

[0004] Patent No. 6639634

[0005] However, the inventors have found that when evaluating the effects of a drug using a three-dimensional cellular tissue, the manifestation of the drug's efficacy may not be clearly detected. Therefore, an object of the present invention is to provide a three-dimensional cellular tissue that allows the effects of a drug to be clearly confirmed.

[0006] The present invention includes the following aspects: [1] A method for producing a three-dimensional cellular tissue, comprising: (a) obtaining a stromal cell-containing mixture containing stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (b) gelling the stromal cell-containing mixture to obtain a first gel composition containing stromal cells; (c) obtaining a target cell-containing mixture containing target cells, a second cationic substance, a second extracellular matrix component, and a second polyelectrolyte; (d) placing the target cell-containing mixture in contact with the first gel composition containing stromal cells; (e) gelling the target cell-containing mixture to obtain a second gel composition containing target cells; and (f) incubating the first gel composition containing stromal cells and the second gel composition containing the target cells to obtain a three-dimensional cellular tissue, wherein the proportion of the total volume of the target cells to the volume of the second gel composition containing the target cells in the three-dimensional cellular tissue is 60% by volume or more. [2] The method of [1] further comprises, after step (e) and before step (f), the step of placing a stromal cell-containing mixture containing the stromal cells, the first cationic substance, the first extracellular matrix component, and the first polyelectrolyte in contact with a second gel-like composition containing the target cells, allowing it to gel to form a third gel-like composition containing stromal cells, and placing the second gel-like composition containing the target cells between the first and third gel-like compositions containing the stromal cells. [3] The method of [1] or [2], wherein the first gel-like composition containing stromal cells comprises a gel-like component formed by gelling at least one selected from the group consisting of an extracellular matrix component, agarose, pectin, and fibrin monomer. [4] The method of [3], wherein the gel-like component is a fibrin gel formed by gelling fibrin monomer, and step (b) of obtaining the first gel-like composition containing stromal cells comprises the step of mixing thrombin and fibrinogen with the stromal cell-containing mixture. [5] The manufacturing method described in [4], wherein step (b) of obtaining a first gel-like composition containing stromal cells includes the steps of adding thrombin to the stromal cell-containing mixture and adding fibrinogen to the stromal cell-containing mixture to which thrombin has been added, thereby forming a fibrin gel and gelling the stromal cell-containing mixture.[6] The manufacturing method according to any one of [1] to [5], wherein the second gel-like composition containing the target cells comprises a gel-like component formed by gelling at least one selected from the group consisting of extracellular matrix components, agarose, pectin, and fibrin monomer. [7] The manufacturing method according to [6], wherein the gel-like component is a fibrin gel formed by gelling fibrin monomer, and step (e) of obtaining the second gel-like composition containing the target cells comprises mixing thrombin and fibrinogen with the target cell-containing mixture. [8] The manufacturing method according to [7], wherein step (e) of obtaining the second gel-like composition containing the target cells comprises adding thrombin to the target cell-containing mixture, and adding fibrinogen to the target cell-containing mixture to which thrombin has been added, thereby forming a fibrin gel and gelling the target cell-containing mixture. [9] The manufacturing method according to any one of [1] to [8], wherein the first and second extracellular matrix components are selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrin, proteoglycan, and combinations thereof.

[10] The manufacturing method according to any one of [1] to [9], wherein the target cells are cancer cells.

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

[10] , wherein the first cationic substance and the second cationic substance are the same, the first extracellular matrix component and the second extracellular matrix component are the same, and the first polyelectrolyte is the same as the second polyelectrolyte.

[12] A three-dimensional cellular tissue, wherein a target cell compartment containing target cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte is arranged in contact with an interstitial cell compartment containing interstitial cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte, and wherein the proportion of the total volume of the target cells to the volume of the target cell compartment is 60% by volume or more.

[13] The three-dimensional cellular tissue according to

[12] , wherein the target cells are cancer cells.

[14] A method for evaluating a test substance, comprising the steps of contacting a test substance with the three-dimensional cellular tissue according to

[12] or

[13] , and evaluating the effect of the test substance on the target cells.

[15] A method for detecting the effect of a test substance, comprising the steps of contacting a test substance with the three-dimensional cellular tissue described in

[12] or

[13] and detecting the effect of the test substance on the target cells.

[0007] According to the present invention, it is possible to provide a three-dimensional cellular tissue in which the manifestation of medicinal effects can be clearly confirmed.

[0008] 1A and 1B are schematic cross-sectional views of a three-dimensional cellular tissue according to one embodiment of the present invention, and 1C are schematic cross-sectional views of a three-dimensional cellular tissue according to another embodiment of the present invention.

[0009] [Method for Producing Three-Dimensional Cellular Tissue] In one embodiment, the present invention provides a method for producing a three-dimensional cellular tissue, the method comprising: (a) obtaining a stromal cell-containing mixture containing stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (b) gelling the stromal cell-containing mixture to obtain a first gel-like composition containing stromal cells; (c) obtaining a target cell-containing mixture containing target cells, a second cationic substance, a second extracellular matrix component, and a second polyelectrolyte; (d) placing the target cell-containing mixture in contact with the first gel-like composition containing stromal cells; (e) gelling the target cell-containing mixture to obtain a second gel-like composition containing target cells; and (f) incubating the first gel-like composition containing stromal cells and the second gel-like composition containing the target cells to obtain a three-dimensional cellular tissue, wherein the total volume of the target cells accounts for 60% or more by volume of the second gel-like composition containing the target cells.

[0010] As described later in the Examples, the manufacturing method of this embodiment allows the production of three-dimensional cellular tissues in which the manifestation of drug efficacy can be clearly confirmed. As described later in the Examples, the three-dimensional cellular tissues produced by the manufacturing method of this embodiment allow for the reproducibility of, for example, evaluation of anticancer drug sensitivity, evaluation of the cancer microenvironment, evaluation of the hypoxic environment, evaluation of cancer-associated fibroblasts, and evaluation of drugs targeting vascular cells. Therefore, the three-dimensional cellular tissues produced by the manufacturing method of this embodiment can be used for screening anticancer drugs, etc.

[0011] As used herein, the phrase "the expression of medicinal effects can be clearly confirmed" means that the three-dimensional cellular tissue produced by the production method of this embodiment has a greater effect on drugs than conventional three-dimensional cellular tissues. The effect on drugs is not particularly limited, and examples thereof include cell viability, cell proliferation, and gene expression.

[0012] Further, examples of conventional three-dimensional cell tissues include three-dimensional cell tissues in which the total volume of the target cells accounts for less than 60% by volume of the volume of a gel composition containing the target cells, and 2D cultures of the target cells.

[0013] As used herein, "three-dimensional cellular tissue" refers to a three-dimensional assembly of cells. Applications of three-dimensional cellular tissue include, but are not limited to, biological tissue models and solid cancer models. Examples of biological tissue models include models of skin, hair, bone, cartilage, teeth, cornea, blood vessels, lymphatic vessels, heart, liver, pancreas, nerves, esophagus, etc. Examples of solid cancer models include models of stomach cancer, esophageal cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, and liver cancer, etc.

[0014] Furthermore, there are no particular limitations on the form of the three-dimensional cell tissue. For example, it may be a three-dimensional cell tissue formed by culturing cells inside a container such as a cell culture insert, a three-dimensional cell tissue formed by culturing cells inside a scaffold composed of natural biopolymers such as collagen or synthetic polymers, a cell aggregate (spheroid), or a sheet-like cell structure.

[0015] First, in step (a), a stromal cell-containing mixture is obtained, which contains stromal cells, cationic substances, extracellular matrix components, and polyelectrolytes. The stromal cells, cationic substances, extracellular matrix components, and polyelectrolytes may be mixed in an aqueous solvent. Examples of aqueous solvents include, but are not limited to, water, buffer solutions, and culture media.

[0016] (Interstitial cells) In this specification, stromal cells refer to cells that constitute the support tissue of epithelial cells. Examples of stromal cells used in this embodiment include fibroblasts, immune cells, vascular endothelial cells, and smooth muscle cells. Examples of immune cells include lymphocytes, neutrophils, and macrophages. Interstitial cells are essential for maintaining tissue and also play important roles in inflammatory responses, wound healing responses, and the like. In the present invention, stromal cells can maintain target cells.

[0017] The origin of the stromal cells is not particularly limited, and cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats can be used.

[0018] (Cationic Substance) Any positively charged substance can be used as the cationic substance, as long as it does not adversely affect the growth of interstitial cells or the formation of interstitial cell aggregates, as described below. Examples of cationic substances include, but are not limited to, cationic buffers such as Tris-hydrochloric acid, Tris-maleic acid, Bis-Tris, and HEPES, as well as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, and polyarginine. Among these, cationic buffers are preferred, and Tris-hydrochloric acid is more preferred.

[0019] The concentration of the cationic substance in the stromal cell-containing mixture in step (a) is not particularly limited, as long as it does not adversely affect the growth of stromal cells and the formation of stromal cell aggregates. The concentration of the cationic substance used in this embodiment is preferably 10 to 100 mM, and may be, for example, 20 to 90 mM, for example, 30 to 80 mM, for example, 40 to 70 mM, or for example, 45 to 60 mM.

[0020] When a cationic buffer is used as the cationic substance, the pH of the cationic buffer is not particularly limited as long as it does not adversely affect the growth of stromal cells and the formation of stromal cell aggregates. The pH of the cationic buffer used in this embodiment is preferably 6.0 to 8.0. For example, the pH of the cationic buffer used in this embodiment may be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The pH of the cationic buffer used in this embodiment is more preferably 7.2 to 7.6, and even more preferably about 7.4.

[0021] (Extracellular matrix components) Any component constituting the extracellular matrix (ECM) can be used as the extracellular matrix component, as long as it does not adversely affect the growth of interstitial cells and the formation of interstitial cell aggregates described below. Examples of extracellular matrix components include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, and combinations thereof. The extracellular matrix component may be a modified or variant of the above-mentioned components. One type of extracellular matrix component may be used alone, or two or more types may be used in combination.

[0022] Examples of proteoglycans include chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans. Of these, collagen, laminin, or fibronectin is preferred as an extracellular matrix component, with collagen being particularly preferred.

[0023] The total content of extracellular matrix components in the stromal cell-containing mixture in step (a) is not particularly limited, as long as it does not adversely affect the growth of stromal cells and the formation of stromal cell aggregates. The total content may be 0.005 mg / mL to 1.5 mg / mL, 0.005 mg / mL to 1.0 mg / mL, 0.01 mg / mL to 1.0 mg / mL, 0.025 mg / mL to 1.0 mg / mL, or 0.025 mg / mL to 0.1 mg / mL. The extracellular matrix components can be used by dissolving them in an appropriate solvent. Examples of solvents include, but are not limited to, water, buffer solutions, and acetic acid. Of these, buffer solutions and acetic acid are preferred.

[0024] (Fragmented Extracellular Matrix Components) The extracellular matrix components may be fragmented extracellular matrix components (hereinafter, sometimes referred to as "fragmented extracellular matrix components"). Fragmented extracellular matrix components can be obtained by fragmenting the above-mentioned extracellular matrix components. "Fragmentation" means breaking down aggregates of extracellular matrix molecules into smaller sizes. Fragmentation may be performed under conditions that cleave the bonds within the extracellular matrix molecules, or under conditions that do not cleave the bonds within the extracellular matrix molecules. The fragmented extracellular matrix components may include defibrated extracellular matrix components, which are components obtained by defibrating the above-mentioned extracellular matrix components by applying a physical force. Defibration is one form of fragmentation, and is performed, for example, under conditions that do not cleave the bonds within the extracellular matrix molecules.

[0025] The method for fragmenting extracellular matrix components is not particularly limited. Examples of methods for defibrating extracellular matrix components include applying physical force using an ultrasonic homogenizer, agitation homogenizer, or high-pressure homogenizer. When using an agitation homogenizer, the extracellular matrix components may be homogenized directly or in an aqueous medium such as physiological saline. Furthermore, by adjusting the homogenization time or number of times, it is possible to obtain millimeter-sized or nanometer-sized defibrated extracellular matrix components. Defibrated extracellular matrix components can also be obtained by defibrating the material through repeated freezing and thawing.

[0026] The fragmented extracellular matrix component may contain at least a defibrated extracellular matrix component. Alternatively, the fragmented extracellular matrix component may consist solely of defibrated extracellular matrix components. That is, the fragmented extracellular matrix component may be a defibrated extracellular matrix component. The defibrated extracellular matrix component preferably contains a defibrated collagen component (hereinafter sometimes referred to as a defibrated collagen component). The defibrated collagen component preferably maintains the triple helix structure derived from collagen. By dispersing the fragmented collagen component in an aqueous medium, it becomes more likely to come into contact with cells in the aqueous medium, which can promote the formation of three-dimensional cellular tissue.

[0027] Examples of the shape of the fragmented extracellular matrix components include fibrous shapes. Fibrous shapes refer to shapes composed of thread-like extracellular matrix components or shapes composed of thread-like extracellular matrix components cross-linked intermolecularly. At least a portion of the fragmented extracellular matrix components may be fibrous. Fibrous extracellular matrix components include thin thread-like materials (hereinafter sometimes referred to as fibrils) formed by the aggregation of multiple thread-like extracellular matrix molecules, thread-like materials formed by the further aggregation of fibrils, and defibrillated versions of these thread-like materials. In fibrous extracellular matrix components, the RGD sequence is preserved without being destroyed, allowing them to function more effectively as scaffolds for cell adhesion.

[0028] 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, from the viewpoint of facilitating the formation of thick tissue, 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, 22 μm or more and 400 μm or less, or 100 μm or more and 400 μm or less. In another embodiment, from the viewpoint of facilitating stable tissue formation and achieving even better redispersibility, 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 as a whole is within the above-mentioned numerical range. Specifically, it is preferable that the average length of 95% of the fragmented extracellular matrix components is within the above-mentioned range. The fragmented extracellular matrix components are preferably fragmented collagen components having an average length within the above-mentioned range, and more preferably defibrated collagen components having an average length within the above-mentioned range.

[0029] 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, and more preferably a defibrated collagen component having an average diameter within the above range.

[0030] The above-mentioned ranges of average length and average diameter have been optimized from the viewpoint of forming three-dimensional cellular tissue, and therefore, after the drying process described below, it is desirable that the average length or average diameter falls within the above-mentioned ranges when the fragmented extracellular matrix components are resuspended in an aqueous medium to form tissue.

[0031] The average length and average diameter of fragmented extracellular matrix components can be determined by measuring individual fragmented extracellular matrix components using an optical microscope and analyzing the images. As used herein, "average length" refers to the arithmetic mean value of the longitudinal length of the measured sample, and "average diameter" refers to the arithmetic mean value of the length of the measured sample in the direction perpendicular to the longitudinal direction.

[0032] At least a portion of the fragmented extracellular matrix components may be inter- or intramolecularly cross-linked. The fragmented extracellular matrix components may be cross-linked within the molecules that make up the fragmented extracellular matrix components, or may be cross-linked between the molecules that make up the fragmented extracellular matrix components.

[0033] Examples of the crosslinking method include physical crosslinking using heat, ultraviolet light, radiation, etc., and chemical crosslinking using a crosslinking agent or an enzyme reaction, etc., but the method is not particularly limited. The crosslinking (i.e., physical crosslinking and chemical crosslinking) may be crosslinking via a covalent bond.

[0034] When the extracellular matrix component includes a collagen component, crosslinks may be formed between triple helical structures of collagen molecules, or between collagen fibrils formed by the collagen molecules. Crosslinking may be thermal crosslinking (i.e., thermal crosslinking). Thermal crosslinking can be performed, for example, by heat treatment under reduced pressure using a vacuum pump. When thermal crosslinking of a collagen component is performed, the extracellular matrix component may be crosslinked by forming a peptide bond (-NH-CO-) between an amino group of the collagen molecule and a carboxy group of the same or another collagen molecule.

[0035] Extracellular matrix components can also be crosslinked using a crosslinking agent. The crosslinking agent may be, for example, one capable of crosslinking carboxyl groups with amino groups, or one capable of crosslinking amino groups with each other. Aldehyde-based, carbodiimide-based, epoxide-based, and imidazole-based crosslinking agents are preferred from the standpoints of economy, safety, and ease of use. Specific examples of crosslinking agents include water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide sulfonate.

[0036] The quantitative determination of the degree of crosslinking can be appropriately selected depending on the type of extracellular matrix component, the crosslinking means, etc. The degree of crosslinking may be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, or 30% or less, 20% or less, or 15% or less. The upper and lower limits of the degree of crosslinking can be arbitrarily combined. For example, the degree of crosslinking may be 1% or more and 30% or less, 2% or more and 20% or less, 4% or more and 15% or less, 8% or more and 15% or less, or 12% or more and 15% or less. A degree of crosslinking within the above range allows the extracellular matrix molecules to be adequately dispersed and also provides good redispersibility after dry storage.

[0037] When amino groups in extracellular matrix components are used for crosslinking, the degree of crosslinking can be quantified by the TNBS method described in, for example, Tatara Y., et al., Chondroitin sulfate cluster of epiphycan from salmon nasal cartilage defines binding specificity to collagens, Glycobiology, 25 (5), 557-569, 2015. The degree of crosslinking measured by the TNBS method is preferably within the above range. The degree of crosslinking measured by the TNBS method is the proportion of amino groups used for crosslinking among the amino groups in the extracellular matrix.

[0038] The degree of crosslinking may be calculated by quantifying the number of 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 measured by the TBO method may be within the above-mentioned range.

[0039] The content of the fragmented extracellular matrix components in the extracellular matrix-containing composition may be 1% by mass or more, 3% by mass or more, 10% by mass or more, 20% by mass or more, 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, 90% by mass or more, 95% by mass or more, or 98% by mass or more, based on the total amount of the extracellular matrix-containing composition, and may be 99% by mass or less, 95% by mass or less, or 90% by mass or less. The upper and lower limits of the content of the fragmented extracellular matrix components in the extracellular matrix-containing composition can be arbitrarily combined. For example, the content of the fragmented extracellular matrix components in the extracellular matrix-containing composition may be 1% by mass or more and 99% by mass or less, 3% by mass or more and 95% by mass or less, or 10% by mass or more and 90% by mass or less.

[0040] (Polyelectrolyte) As used herein, the term "polyelectrolyte" refers to a polymer having a dissociable functional group in the polymer chain. Any polyelectrolyte can be used in the present embodiment as long as it does not adversely affect the growth of interstitial cells and the formation of interstitial cell aggregates. Examples of polyelectrolytes include, but are not limited to, glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate and 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, polyacrylic acid, and combinations thereof. The polyelectrolyte may be a derivative of any of the above. These polyelectrolytes may be used alone or in combination of two or more.

[0041] The polyelectrolyte is preferably a glycosaminoglycan, and among these, heparin, chondroitin sulfate, or dermatan sulfate is preferred, with heparin being particularly preferred.

[0042] The concentration of the polyelectrolyte in the stromal cell-containing mixture in step (a) is not particularly limited, as long as it does not adversely affect the growth of stromal cells and the formation of stromal cell aggregates. Unlike extracellular matrix components, polyelectrolytes are effective at any concentration as long as it is below the solubility limit and does not inhibit the effects of the extracellular matrix components. The concentration of the polyelectrolyte is preferably 0.005 mg / mL or more, and may be 0.005 mg / mL to 1.0 mg / mL, 0.01 mg / mL to 1.0 mg / mL, 0.025 mg / mL to 1.0 mg / mL, or 0.025 mg / mL to 0.1 mg / mL.

[0043] The polymer electrolyte may be dissolved in a suitable solvent. Examples of the solvent include, but are not limited to, water and a buffer solution. When a cationic buffer solution is used as the cationic substance, the polymer electrolyte may be dissolved in the cationic buffer solution.

[0044] The blending ratio (final concentration ratio) of the polyelectrolyte to the extracellular matrix component in the stromal cell-containing mixture in step (a) is preferably 1:2 to 2:1, may be 1:1.5 to 1.5:1, or may be 1:1.

[0045] In step (a), the stromal cells, cationic substance, extracellular matrix component, and polyelectrolyte can be mixed in an appropriate container such as a dish, a tube, a flask, a bottle, a well plate, a cell culture insert, etc. Mixing of these components may also be carried out in the container used in step (b).

[0046] The stromal cell-containing mixture in step (a) may contain other components in addition to the stromal cells, cationic substances, extracellular matrix components, and polyelectrolytes, such as a gelling agent and a cell culture medium required to obtain a gel composition in step (b).

[0047] (Gelling Agent) Examples of gelling agents include extracellular matrix components, agarose, pectin, a combination of fibrinogen and thrombin, etc. The gelling agent may be contained in advance in the stromal cell-containing mixture in step (a), or may be added to the stromal cell-containing mixture in step (a) in step (b) described below.

[0048] After step (a), in step (b), the stromal cell-containing mixture obtained in step (a) is gelled to obtain a gel composition containing stromal cells. The gelling method varies depending on the gelling agent used, and may be, for example, placing the stromal cell-containing mixture obtained in step (a) under gelling conditions. Alternatively, a gelling agent may be added to the stromal cell-containing mixture obtained in step (a) and then placing the mixture under gelling conditions.

[0049] For example, when an extracellular matrix component is used as the gelling agent, gelation conditions include allowing the stromal cell-containing mixture obtained in step (a) to stand at about 37°C. As a result, the extracellular matrix component contained in the stromal cell-containing mixture in step (a) gels, yielding a gel composition containing stromal cells. Alternatively, an extracellular matrix component may be further added to the stromal cell-containing mixture obtained in step (a), and the mixture may be allowed to stand at about 37°C to gel.

[0050] Furthermore, when agarose is used as a gelling agent, agarose may be added to the stromal cell-containing mixture obtained in step (a), the agarose may be dissolved at a temperature equal to or higher than the melting point of the agarose used, and then the mixture may be allowed to stand at a temperature equal to or lower than the solidification point of the agarose used to cause gelation.

[0051] When pectin is used as a gelling agent, the pectin may be added to the stromal cell-containing mixture obtained in step (a), resulting in gelation of the pectin by divalent ions such as calcium ions contained in the stromal cell-containing mixture, thereby obtaining a gel composition.

[0052] Fibrinogen and thrombin may also be used as gelling agents. Thrombin, a type of serine protease, cleaves fibrinogen to form fibrin monomers. Fibrin monomers polymerize with each other under the action of calcium ions to form poorly soluble fibrin polymers. In vivo, fibrin polymers are cross-linked by the action of factor XIII, a fibrin stabilizing factor, to form mesh-like fibers called stabilized fibrin, which causes blood coagulation. In this specification, a gel composition gelled by fibrin polymers may be referred to as a fibrin gel.

[0053] That is, step (b) of obtaining a gel composition containing stromal cells may include a step of mixing thrombin and fibrinogen with the stromal cell-containing mixture obtained in step (a).The gel composition containing stromal cells in step (b) may contain fibrin gel as a gel component.

[0054] In this case, it is preferable that step (b) of obtaining a gel composition containing stromal cells comprises step (b1) of adding thrombin to the stromal cell-containing mixture obtained in step (a), and step (b2) of adding fibrinogen to the stromal cell-containing mixture to which thrombin has been added, thereby forming a fibrin gel and gelling the stromal cell-containing mixture.

[0055] When fibrinogen and thrombin are used as gelling agents, the fibrinogen concentration in the stromal cell-containing mixture in step (b) is preferably 0.5 mg / mL or more and 25 mg / mL or less. A fibrinogen concentration of 0.5 mg / mL or more facilitates gelation when mixed with thrombin. Furthermore, a fibrinogen concentration of 25 mg / mL or less facilitates dissolution in the stromal cell-containing mixture. Furthermore, it is preferred that thrombin be dissolved or dispersed in the stromal cell-containing mixture in step (b).

[0056] If the stromal cell-containing mixture contains a substance with anticoagulant properties (typically, heparin is selected as the polyelectrolyte), it is thought that fibrin will not polymerize. For this reason, a substance with anticoagulant properties and fibrin are not usually used simultaneously. In contrast, in this embodiment, fibrin polymerization is not inhibited, although it is unclear whether this is due to the combination of materials or the low concentration.

[0057] The inventors also found that, depending on the conditions, adding fibrinogen first to the stromal cell-containing mixture obtained in step (a) can result in gelation by itself. They speculate that this is due to the fibrinogen being cleaved by some protease contained in the stromal cell-containing mixture obtained in step (a) to form fibrin monomers. Therefore, when mixing thrombin and fibrinogen with the stromal cell-containing mixture obtained in step (a), it is preferable to mix the thrombin first and then the fibrinogen. Alternatively, a gel composition containing stromal cells can be obtained by adding only fibrinogen as a gelling agent in step (b).

[0058] Thus, the gel composition containing stromal cells formed in step (b) may contain a gel component formed by gelling at least one of extracellular matrix components, agarose, pectin, and fibrin monomers.

[0059] Next, in step (c), a target cell-containing mixture containing the target cells, cationic substances, extracellular matrix components, and polyelectrolytes is obtained.

[0060] (Target Cells) As used herein, target cells refer to cells that are the target for maintenance using stromal cells. The target cells are not particularly limited, and cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats can be used. The site of origin of the target cells is also not particularly limited, and they may be somatic cells derived from bone, muscle, internal organs, nerves, brain, skin, blood, etc., or may be germ cells or cancer cells.

[0061] Examples of somatic cells derived from blood include immune cells such as lymphocytes, neutrophils, macrophages, and dendritic cells, and examples of cancer cells include gastric cancer, esophageal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell cancer, and liver cancer.

[0062] Furthermore, the target cells may be pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells), or tissue stem cells.

[0063] The target cells may be primary cells or cultured cells such as passaged cells and cell line cells. Furthermore, one type of cell may be used alone, or multiple types may be used in combination. Furthermore, the target cells may be subjected to gene editing, gene transfer, or the like. For example, the target cells may be cancer cells in which the expression level of a specific protein has been increased by gene transfer.

[0064] The cationic substance, extracellular matrix component, and polyelectrolyte used in step (c) include those used in step (a). The cationic substance, extracellular matrix component, and polyelectrolyte used in step (c) may be the same as the cationic substance, extracellular matrix component, and polyelectrolyte used in step (a), or may be different from the cationic substance, extracellular matrix component, and polyelectrolyte used in step (a).

[0065] In step (c), the target cells, cationic substance, extracellular matrix component, and polyelectrolyte can be mixed in an appropriate container such as a dish, tube, flask, bottle, well plate, or cell culture insert, or the like. Mixing of these components may also be performed in the container used in step (d).

[0066] The target cell-containing mixture in step (c) may contain other components in addition to the target cells, cationic substances, extracellular matrix components, and polyelectrolytes, such as a gelling agent and cell culture medium required to obtain a gel composition containing the target cells in step (e).

[0067] Examples of gelling agents include extracellular matrix components, agarose, pectin, a combination of fibrinogen and thrombin, etc. The gelling agent may be contained in advance in the target cell-containing mixture in step (c), or may be added to the target cell-containing mixture in step (c) in step (e) described below.

[0068] Subsequently, in step (d), the target cell-containing mixture is placed in contact with a gel composition containing stromal cells. The method of placement is not particularly limited as long as the target cells are placed in contact with the gel composition containing stromal cells, and the target cells may be placed on or in the gel composition containing stromal cells.

[0069] When target cells are placed in a gel composition containing stromal cells, it is preferable to layer the stromal cell-containing mixture obtained in the same manner as in step (a) on the gel composition containing target cells after step (e) described below, and then gel the stromal cell-containing mixture layered on the gel composition containing target cells in the same manner as in step (b), thereby layering the gel composition containing stromal cells, i.e., the third gel composition, on the gel composition containing target cells. That is, the second gel composition containing target cells may be placed between the first gel composition containing stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte, and the third gel composition containing stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte.

[0070] After step (d), in step (e), the target cell-containing mixture obtained in step (c) is gelled to obtain a gel composition containing the target cells. The gelling method varies depending on the gelling agent used, and may be, for example, placing the target cell-containing mixture obtained in step (c) under gelling conditions. Alternatively, a gelling agent may be added to the target cell-containing mixture obtained in step (c) and then placing it under gelling conditions.

[0071] The gelling conditions for obtaining a gel composition containing target cells in step (e) are the same as the gelling conditions for obtaining a gel composition containing stromal cells in step (b) above, except that target cells are used instead of stromal cells.

[0072] In step (d), the target cell-containing mixture placed in contact with the gel composition containing interstitial cells is prevented from migrating or expanding from the position of the gel composition containing interstitial cells by gelling the target cell-containing mixture, thereby allowing the target cells to be maintained in a predetermined position in the three-dimensional cellular tissue.

[0073] Subsequently, in step (f), the gel composition containing stromal cells obtained in step (b) and the gel composition containing target cells obtained in step (e) are incubated to obtain a three-dimensional cellular tissue. The incubation and culture time for the gel composition containing stromal cells and the gel composition containing target cells to obtain a three-dimensional cellular tissue may be 5 minutes to 72 hours. Step (f) promotes adhesion between the interstitial cells contained in the gel composition containing stromal cells and between the target cells contained in the gel composition containing target cells, resulting in a stable three-dimensional cellular tissue. Furthermore, since the migration or expansion of the target cells from their positions in the gel composition containing stromal cells is inhibited, the target cells can be maintained in their predetermined positions in the three-dimensional cellular tissue. This allows for the construction of organs that require more structural three-dimensional control. Furthermore, since the thickness of the target cells can be maintained, large tissues can be cultured over the long term.

[0074] The culture of the stromal cells and target cells in step (f) can be carried out under culture conditions suitable for the cells to be cultured. Those skilled in the art can select an appropriate culture medium depending on the type of stromal cells and target cells and the desired function. The culture medium is not particularly limited, but examples include DMEM, EMEM, MEMα, RPMI-1640, McCoy's 5A, Ham's F-12, etc., and media containing these media supplemented with approximately 1 to 20% by volume of serum. Examples of serum include bovine serum (CS), fetal bovine serum (FBS), and fetal horse serum (HBS). Various conditions of the culture environment, such as the temperature and atmospheric composition, may also be adjusted to conditions suitable for the stromal cells and target cells to be cultured.

[0075] The container used in step (f) may be the same as the container used in step (b). In step (f), the container used in step (b) may be used as is, or the contents may be transferred to another container.

[0076] During the culture of stromal cells and target cells, a substance may be added to the medium to suppress deformation of the resulting three-dimensional cell tissue (e.g., tissue contraction, detachment of tissue edges, etc.) Examples of such a substance include, but are not limited to, Y-27632, a Rho-associated coiled-coil forming kinase / Rho-associated kinase (ROCK) inhibitor.

[0077] In the three-dimensional cellular tissue produced by the production method of this embodiment, the ratio of the total volume of the target cells to the volume of the gel composition containing the target cells (in other words, the volume of the target cell compartment) is 60% by volume or more. The ratio of the total volume of the target cells to the volume of the target cell compartment may be 70% by volume or more, 80% by volume or more, or even 90% by volume or more. As will be described later in the Examples, when the total volume of the target cells is within the above range, a three-dimensional cellular tissue is obtained in which the manifestation of medicinal effects can be clearly confirmed. The upper limit of the ratio of the total volume of the target cells to the volume of the target cell compartment is not particularly limited, but an example thereof is 99.9% by volume.

[0078] As long as the total volume of the target cells reaches 60% or more by volume of the target cell compartment by continuing incubation of the three-dimensional cellular tissue, the total volume of the target cells may be less than 60% by volume of the target cell compartment when the target cells are seeded. The three-dimensional cellular tissue of this embodiment is completed when the total volume of the target cells reaches 60% or more by volume of the target cell compartment. If the number of seeded control cells is low, the total volume of the target cells will not reach 60% by volume of the target cell compartment even if incubation of the three-dimensional cellular tissue is continued.

[0079] (Cell aggregate) The production method of this embodiment may further include, prior to step (b) of obtaining a gel-like composition containing stromal cells, step (a') of applying an external force to the stromal cell-containing mixture obtained in step (a) to obtain a stromal cell aggregate containing stromal cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte, and in step (b) of obtaining a gel-like composition containing stromal cells, the stromal cell aggregate obtained in step (a') may be gelled instead of the stromal cell-containing mixture obtained in step (a) to obtain a gel-like composition containing stromal cells.

[0080] In this case, the method for producing a three-dimensional cellular tissue of this embodiment includes the steps of: (a) obtaining a stromal cell-containing mixture containing stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (a') applying an external force to the stromal cell-containing mixture to obtain a stromal cell aggregate containing first stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (b') gelling the stromal cell aggregate to obtain a first gel-like composition containing stromal cells; (c) obtaining a target cell-containing mixture containing target cells, a second cationic substance, a second extracellular matrix component, and a second polyelectrolyte; (d) placing the target cell-containing mixture in contact with the first gel-like composition containing stromal cells; (e) gelling the target cell-containing mixture to obtain a second gel-like composition containing target cells; and (f) incubating the first gel-like composition containing stromal cells and the second gel-like composition containing the target cells to obtain a three-dimensional cellular tissue.

[0081] Furthermore, the manufacturing method of this embodiment may further comprise a step (c') of applying an external force to the target cell-containing mixture obtained in step (c) to obtain a target cell aggregate comprising the target cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte, prior to step (d) of placing the target cell-containing mixture so that it is in contact with a gel-like composition comprising the stromal cells; in step (d) of placing the target cell-containing mixture so that it is in contact with a gel-like composition comprising the stromal cells, the target cell aggregate obtained in step (c') may be placed in contact with the gel-like composition comprising the stromal cells instead of the target cell-containing mixture; and in step (e) of obtaining a gel-like composition comprising the target cells, the target cell aggregate obtained in step (c') may be gelled instead of the target-containing mixture obtained in step (c), to obtain a gel-like composition comprising the target cells.

[0082] In this case, the method for producing a three-dimensional cellular tissue of this embodiment includes the steps of: (a) obtaining a stromal cell-containing mixture containing stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (b) gelling the stromal cell-containing mixture to obtain a first gel-like composition containing stromal cells; (c) obtaining a target cell-containing mixture containing target cells, a second cationic substance, a second extracellular matrix component, and a second polyelectrolyte; (c') applying an external force to the target cell-containing mixture to obtain a target cell aggregate containing the target cells, the second cationic substance, the second extracellular matrix component, and the second polyelectrolyte; (d') placing the target cell aggregate in contact with the first gel-like composition containing stromal cells; (e') gelling the target cell aggregate to obtain a second gel-like composition containing target cells; and (f) incubating the first gel-like composition containing the target cells and the second gel-like composition containing the target cells to obtain a three-dimensional cellular tissue.

[0083] Furthermore, the manufacturing method of this embodiment further comprises, before step (b) of obtaining a gel-like composition containing stromal cells, step (a') of applying an external force to the stromal cell-containing mixture obtained in step (a) to obtain a stromal cell aggregate containing stromal cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte; in step (b) of obtaining a gel-like composition containing stromal cells, the stromal cell aggregate obtained in step (a') is gelled instead of the stromal cell-containing mixture obtained in step (a) to obtain a gel-like composition containing stromal cells; and further, before step (d) of placing the target cell-containing mixture in contact with the gel-like composition containing stromal cells, applying an external force to the target cell-containing mixture obtained in step (c). and obtaining a target cell aggregate containing target cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte; in step (d) of placing the target cell-containing mixture in contact with a gel composition containing the stromal cells, the target cell aggregate obtained in step (c') is placed in contact with the gel composition containing the stromal cells instead of the target cell-containing mixture; and in step (e) of obtaining a gel composition containing target cells, the target cell aggregate obtained in step (c') is gelled instead of the target-containing mixture obtained in step (c) to obtain a gel composition containing target cells.

[0084] In this case, the method for producing a three-dimensional cellular tissue of this embodiment includes the steps of: (a) obtaining a stromal cell-containing mixture containing target cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (a') obtaining a stromal cell aggregate containing stromal cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (b') gelling the stromal cell aggregate to obtain a first gel composition containing stromal cells; and (b') obtaining a target cell-containing mixture containing target cells, a second cationic substance, a second extracellular matrix component, and a second polyelectrolyte. (c), a step (c') of applying an external force to the mixture containing the target cells to obtain a target cell aggregate containing the target cells, a second cationic substance, a second extracellular matrix component, and a second polymer electrolyte, a step (d') of placing the target cell aggregate so that it is in contact with a first gel-like composition containing the stromal cells, a step (e') of gelling the target cell aggregate to obtain a second gel-like composition containing the target cells, and a step (f) of incubating the first gel-like composition containing the target cells and the second gel-like composition containing the target cells to obtain a three-dimensional cellular tissue.

[0085] As used herein, the term "cell aggregate" refers to a structure formed by cells gathering together. Cell aggregates also include cell precipitates obtained by centrifugation, filtration, or the like. In one embodiment, the cell aggregate is a slurry-like viscous body. The term "slurry-like viscous body" refers to a gel-like cell aggregate such as that described in Akihiro Nishiguchi et al., Cell-cell crosslinking by bio-molecular recognition of heparin-based layer-by-layer nanofilms, Macromol Biosci., 15 (3), 312-317, 2015.

[0086] The cell aggregates can be formed by placing the stromal cell-containing mixture obtained in step (a) or the target cell-containing mixture obtained in step (c) in an appropriate container and allowing it to stand, or by placing the stromal cell-containing mixture obtained in step (a) or the target cell-containing mixture obtained in step (c) in an appropriate container and collecting the cells by, for example, centrifugation, magnetic separation, filtration, or the like to form cell aggregates. That is, the application of an external force in step (a') or step (c') may mean allowing the mixture to stand and allowing gravity to act, or performing centrifugation, magnetic separation, filtration, or the like. When the cells are collected by standing, centrifugation, magnetic separation, filtration, or the like, the liquid portion may or may not be removed.

[0087] The vessel used in step (a') or step (c') may be a culture vessel for cell culture. The culture vessel may be a vessel made of a material and having a shape commonly used for culturing cells or microorganisms. Materials for the culture vessel include, but are not limited to, glass, stainless steel, plastic, etc. Examples of culture vessels include, but are not limited to, dishes, tubes, flasks, bottles, well plates, and cell culture inserts. It is preferable that at least a portion of the vessel is formed from a material that is permeable to liquid but does not allow cells in liquid to pass through. Examples of such vessels include, but are not limited to, cell culture inserts such as Transwell (registered trademark) inserts, Netwell (registered trademark) inserts, Falcon (registered trademark) cell culture inserts, and Millicell (registered trademark) cell culture inserts.

[0088] The conditions for centrifugation are not particularly limited as long as they do not adversely affect the growth of stromal cells and target cells. For example, the stromal cell-containing mixture or the target cell-containing mixture can be placed in a cell culture insert and centrifuged at 10°C and 400 × g for 1 minute to collect the stromal cells or target cells, thereby obtaining a stromal cell aggregate or a target cell aggregate.

[0089] Step (b') of gelling the stromal cell aggregate to obtain a gel composition containing stromal cells can be carried out in the same manner as step (b) of gelling the stromal cell-containing mixture obtained in step (a) to obtain a gel composition.

[0090] Furthermore, step (c') of gelling the target cell aggregate to obtain a gel composition containing the target cells can be carried out in the same manner as step (c) described above, of gelling the target cell-containing mixture obtained in step (c) to obtain a gel composition containing the target cells.

[0091] For example, when an extracellular matrix component is used as a gelling agent, the stromal cell aggregate obtained in step (a') or the target cell aggregate obtained in step (c') may be left to stand at approximately 37°C, or an extracellular matrix component may be further added to the stromal cell aggregate obtained in step (a') or the target cell aggregate obtained in step (c') and then left to stand at approximately 37°C to gel.

[0092] Furthermore, when agarose is used as a gelling agent, agarose may be added to the stromal cell aggregates obtained in step (a') or the target cell aggregates obtained in step (c'), the agarose may be dissolved at a temperature above the melting point of the agarose used, and then the mixture may be allowed to stand at a temperature below the solidification point of the agarose used to gel it.

[0093] Furthermore, when pectin is used as a gelling agent, pectin may be added to the stromal cell mass obtained in step (a') or the target cell mass obtained in step (c'), resulting in gelation of the pectin by divalent ions such as calcium ions contained in the stromal cell-containing mixture or the target cells, thereby obtaining a gel-like composition containing stromal cells or a gel-like composition containing the target cells.

[0094] Alternatively, fibrinogen and thrombin may be used as gelling agents. That is, step (b') of obtaining a gel composition containing stromal cells may include a step of mixing thrombin and fibrinogen with the stromal cell mass obtained in step (a'). The gel composition containing stromal cells in step (b') may contain fibrin gel as a gel component.

[0095] In this case, it is preferable that step (b') of obtaining a gel composition containing stromal cells comprises step (b1') of adding thrombin to the stromal cell aggregate obtained in step (a'), and step (b2') of adding fibrinogen to the stromal cell aggregate to which thrombin has been added, resulting in the formation of a fibrin gel and gelling of the stromal cell-containing mixture.

[0096] Furthermore, step (e') of obtaining a gel composition containing target cells may include a step of mixing thrombin and fibrinogen with the target cell mass obtained in step (c'). The gel composition containing target cells in step (e') may contain fibrin gel as a gel component.

[0097] In this case, it is preferable that step (e') of obtaining a gel composition containing target cells includes step (e1') of adding thrombin to the target cell mass obtained in step (c'), and step (e2') of adding fibrinogen to the target cell mass to which thrombin has been added, resulting in the formation of a fibrin gel and gelling of the mixture.

[0098] Subsequently, in step (f), the gel composition containing the stromal cells obtained in step (b') and the gel composition containing the target cells obtained in step (e') are incubated to obtain a three-dimensional cellular tissue. Step (f) is the same as that described above.

[0099] [Three-dimensional cellular tissue] In one embodiment, the present invention provides a three-dimensional cellular tissue in which a target cell compartment containing target cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte is arranged in contact with an interstitial cell compartment containing interstitial cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte, and the proportion of the total volume of the target cells to the volume of the target cell compartment is 60% by volume or more.

[0100] As will be described later in the Examples, the three-dimensional cellular tissue of this embodiment can be used to clearly confirm the manifestation of drug efficacy. Therefore, the three-dimensional cellular tissue of this embodiment can be used to reproducibly evaluate, for example, anticancer drug sensitivity, cancer microenvironment, hypoxic environment, cancer-associated fibroblasts, and drugs targeting vascular cells. Therefore, the three-dimensional cellular tissue of this embodiment can be used for screening anticancer drugs, etc.

[0101] In the three-dimensional cellular tissue of this embodiment, the total volume of the target cells relative to the volume of the target cell compartment may be 70% by volume or more, 80% by volume or more, or 90% by volume or more. The upper limit of the ratio of the total volume of the target cells to the volume of the target cell compartment is not particularly limited, but may be, for example, 99.9% by volume.

[0102] In the three-dimensional cellular tissue of this embodiment, the interstitial cell compartment may further contain a gel-like component, and the target cell compartment may further contain a gel-like component.

[0103] In the three-dimensional cellular tissue of this embodiment, the stromal cell compartment and the target cell compartment being arranged in contact with each other may mean either that the target cell compartment is arranged on top of the stromal cell compartment, or that the target cells are arranged in the interstitial cell compartment.

[0104] Fig. 1 is a schematic cross-sectional view of a three-dimensional cellular tissue according to one embodiment of the present invention. As shown in Fig. 1, the three-dimensional cellular tissue 1 has a stromal cell compartment 11 and a target cell compartment 12. The target cell compartment 12 is disposed on and in contact with the stromal cell compartment 11.

[0105] Fig. 2 is a schematic cross-sectional view of a three-dimensional cellular tissue according to another embodiment of the present invention. As shown in Fig. 2, the three-dimensional cellular tissue 1 has a stromal cell compartment 11 and a target cell compartment 12. The target cell compartment 12 is embedded within the stromal cell compartment 11. The stromal cell compartment 11 inhibits the target cell compartment 12 from migrating or expanding from a predetermined position, thereby maintaining the target cell compartment at a predetermined position in the three-dimensional cellular tissue.

[0106] In the three-dimensional cellular tissue 1, the inside of the target cell compartment 12 may be a hypoxic environment. The presence of a hypoxic environment can be confirmed, for example, by measuring the expression level of a hypoxia-related marker gene expressed by the target cells in the target cell compartment 12. Examples of methods for measuring the expression level of a hypoxia-related marker gene include quantitative real-time PCR. Examples of hypoxia-related marker genes include APBA3 (MINT3).

[0107] In the three-dimensional cellular tissue of this embodiment, the interstitial cells, target cells, cationic substances, extracellular matrix components, polymer electrolytes, gel-like components, and fragmented extracellular matrix components are the same as those described above.

[0108] [Method for evaluating a test substance and method for detecting its effect] In one embodiment, the present invention provides a method for evaluating a test substance, comprising the steps of contacting a test substance with the above-mentioned three-dimensional cellular tissue and evaluating the effect of the test substance on the target cells.

[0109] In another aspect, the present invention provides a method for detecting the effect of a test substance, comprising the steps of contacting a test substance with the above-mentioned three-dimensional cellular tissue and detecting the effect of the test substance on the target cells.

[0110] As will be described later in the Examples, the use of the above-described three-dimensional cellular tissue allows for clear confirmation of the manifestation of drug efficacy. Therefore, the evaluation method of this embodiment allows for, for example, evaluation of anticancer drug sensitivity, evaluation of the cancer microenvironment, evaluation of a hypoxic environment, evaluation of cancer-associated fibroblasts, evaluation of drugs targeting vascular cells, and the like, to be performed with good reproducibility. Furthermore, the detection method of this embodiment allows for, for example, detection of the efficacy of an anticancer drug on cancer cells, detection of changes in the cancer microenvironment caused by an anticancer drug, detection of the efficacy of an anticancer drug in a hypoxic environment, detection of changes in cancer-associated fibroblasts caused by an anticancer drug, detection of the effect of a drug targeting vascular cells, and the like, to be performed with good reproducibility. Therefore, the evaluation method and detection method of this embodiment can be used for screening anticancer drugs, etc.

[0111] In the evaluation method and detection method of this embodiment, the three-dimensional cellular tissue, interstitial cells, target cells, cationic substances, extracellular matrix components, polymer electrolytes, gel-like components, and fragmented extracellular matrix components are the same as those described above.

[0112] The test substance is not particularly limited, and for example, a natural compound library, a synthetic compound library, or an existing drug library can be used.

[0113] The effect of a test substance on target cells is not particularly limited, and examples thereof include cell viability, cell proliferation, and gene expression.

[0114] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0115] [Materials and Methods] (Cells Constituting the Interstitium) The cells shown in Table 1 below were used as cells constituting the interstitium.

[0116]

[0117] (Subject Cells) The cells shown in Table 2 below were used as subject cells.

[0118]

[0119] (Reagents) The reagents used in the experimental examples described below are shown in Table 3 below.

[0120]

[0121] (Cell Culture Vessel) The cell culture vessels used in the experimental examples described below are shown in Table 4 below.

[0122]

[0123] (Culture Medium) The culture medium used in the experimental examples described below is shown in Table 5 below.

[0124]

[0125] Comparative Example 1 (Production of Three-Dimensional Cellular Tissue) Three-dimensional cellular tissue was produced in Comparative Example 1. NHDF, which are human neonatal dermal fibroblasts, and HUVEC, which are human umbilical vein endothelial cells, were used as stromal cells, and GFP-AsPC-1, a GFP-transfected human pancreatic cancer cell line, was used as cancer cells.

[0126] Preparation of the first interstitial cell compartment: 2 x 10 ６ NHDF and 3 x 10 ４ The HUVECs were suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.05 mg / mL heparin and 0.05 mg / mL collagen.

[0127] The resulting cell suspension was then centrifuged at 1,000 xg for 1 minute at room temperature, and the supernatant was removed. The cells were then resuspended in 50 µL of general-purpose medium, and the entire volume was seeded into a Transwell culture insert.

[0128] Next, the Transwell culture insert was placed in a ２ Incubator (37°C, 5% CO ２ The mixture was left standing in a refrigerator for 24 hours.

[0129] <<Creation of target cell compartment>> 1 x 10 ３ GFP-AsPC-1 was suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.05 mg / mL heparin and 0.05 mg / mL collagen.

[0130] The resulting cell suspension was then centrifuged at 1,000 × g for 1 minute at room temperature, and the supernatant was removed. The cells were then resuspended in 5 μL of general-purpose medium, and the entire amount was seeded onto the center of the Transwell culture insert obtained above.

[0131] Next, the cell culture insert was placed in a ２ Incubator (37°C, 5% CO ２ ) for 2 hours to obtain a cell culture insert on which cancer cells were layered.

[0132] Preparation of a second interstitial cell compartment: 2 x 10 ６ NHDF and 3 x 10 ４The HUVECs were suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.05 mg / mL heparin and 0.05 mg / mL collagen.

[0133] Subsequently, the obtained cell suspension was centrifuged at 1,000 × g for 1 minute at room temperature, and after removing the supernatant, the cells were resuspended in 50 μL of general-purpose medium, and the entire amount was layered in the Transwell culture insert obtained above.

[0134] Next, the cell culture insert was placed in a ２ Incubator (37°C, 5% CO ２ ) for 24 hours or more to obtain a three-dimensional cell tissue of Comparative Example 1.

[0135] [Comparative Example 2] (Production of Three-Dimensional Cellular Tissue) A three-dimensional cellular tissue was produced in Comparative Example 2. The three-dimensional cellular tissue in Comparative Example 2 differed from the three-dimensional cellular tissue in Comparative Example 1 mainly in that fibrinogen and thrombin were used.

[0136] NHDF, which are human neonatal skin fibroblasts, and HUVEC, which are human umbilical vein endothelial cells, were used as stromal cells, and GFP-AsPC-1, a GFP-transfected human pancreatic cancer cell line, was used as cancer cells.

[0137] Preparation of First Stromal Cell Compartment: Thrombin was dissolved in the above-mentioned general-purpose medium to a final concentration of 10 Units / mL to prepare a thrombin solution. Fibrinogen was dissolved in DMEM medium to a final concentration of 10 mg / mL to prepare a fibrinogen solution. Furthermore, a medium was prepared by mixing the fibrinogen solution and the general-purpose medium in a 1:1 (volume ratio) mixture (hereinafter sometimes referred to as "fibrinogen-supplemented general-purpose medium").

[0138] Next, 2 x 10 ６ NHDF and 3 x 10 ４ The HUVECs were suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.05 mg / mL heparin and 0.05 mg / mL collagen.

[0139] The resulting cell suspension was then centrifuged at 1,000 × g for 1 minute at room temperature, the supernatant was removed, and the cells were resuspended in thrombin solution. The thrombin solution containing the cells was then mixed with fibrinogen-supplemented general-purpose medium at a volume ratio of 1:2, and 50 μL of this mixture was seeded into a Transwell culture insert.

[0140] Next, the Transwell culture insert was placed in a ２ Incubator (37°C, 5% CO ２ ) until gelation occurred, to obtain a fibrin gel-containing cell culture insert.

[0141] <<Creation of target cell compartment>> 1 x 10 ３ GFP-AsPC-1 was suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.05 mg / mL heparin and 0.05 mg / mL collagen.

[0142] The resulting cell suspension was then centrifuged at 1,000 × g for 1 minute at room temperature, the supernatant was removed, and the cells were resuspended in thrombin solution. The thrombin solution containing the cells was then mixed with fibrinogen-supplemented general-purpose medium at a volume ratio of 1:2, and 5 μL of this mixed solution was seeded into the center of the fibrin gel-containing cell culture insert obtained above.

[0143] Next, the cell culture insert was placed in a ２ Incubator (37°C, 5% CO ２ ) until gelation occurred, yielding a fibrin gel-containing cell culture insert layered with cancer cells.

[0144] Preparation of a second interstitial cell compartment: 2 x 10 ６ NHDF and 3 x 10 ４ The HUVECs were suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.05 mg / mL heparin and 0.05 mg / mL collagen.

[0145] The resulting cell suspension was then centrifuged at 1,000 × g for 1 minute at room temperature, the supernatant was removed, and the cells were resuspended in thrombin solution. The thrombin solution containing the cells was then mixed with a fibrinogen-supplemented general-purpose medium at a volume ratio of 1:2, and 50 μL of this mixed solution was layered on the fibrin gel-containing cell culture insert seeded with the cancer cells obtained above.

[0146] Next, the cell culture insert was placed in a ２ Incubator (37°C, 5% CO ２ The mixture was left to stand in a container until it gelled, and the three-dimensional cell tissue of Comparative Example 1 was obtained.

[0147] [Comparative Example 3] (Production of three-dimensional cell tissue) 1 × 10 ４ A three-dimensional cell tissue of Comparative Example 3 was obtained by producing a three-dimensional cell tissue in the same manner as in Comparative Example 2, except that GFP-AsPC-1 was used.

[0148] Comparative Example 4 (Production of 2D culture) 2 x 10 ６ GFP-AsPC-1 cells were suspended in 50 μL of general-purpose medium and seeded onto a 24-well plate to obtain the 2D culture of Comparative Example 4.

[0149] [Example 1] (Production of Three-Dimensional Cellular Tissue) A three-dimensional cellular tissue was produced in Example 1. The three-dimensional cellular tissue of Example 1 differed from the three-dimensional cellular tissue of Comparative Example 2 mainly in the concentration of the thrombin solution, the concentration of the fibrinogen solution, and the number of cancer cells in the production of the target cell compartment.

[0150] In preparing the target cell compartment, the final concentration of thrombin in the thrombin solution was 50 Units / mL, the final concentration of fibrinogen in the fibrinogen solution was 50 mg / mL, and 1 × 10 ５ A three-dimensional cell tissue was produced in the same manner as in Comparative Example 2, except that GFP-AsPC-1 was used, and the three-dimensional cell tissue of Example 1 was obtained.

[0151] [Example 2] (Production of three-dimensional cell tissue) 1 x 10 ６ A three-dimensional cell tissue of Example 2 was obtained by producing a three-dimensional cell tissue in the same manner as in Example 1, except that GFP-AsPC-1 was used.

[0152] Experimental Example 1 (Analysis of Three-Dimensional Cellular Tissue) The three-dimensional cellular tissue of Example 1 was cultured using the dedicated medium described above. Subsequently, on the third day after the start of culture, the cells were observed under a microscope, and the number of GFP-AsPC-1 cells was counted based on GFP fluorescence. The percentage of the total volume of the target cells (GFP-AsPC-1) in the volume of the target cell compartment was also measured. More specifically, fibrin and GFP regions were stained using immunohistochemical staining (IHC). Subsequently, cross-sectional analysis was performed at five points per sample, and the volume of the GFP-stained region relative to the volume of the fibrin-stained region was calculated, thereby determining the percentage of the total volume of the target cells in the volume of the target cell compartment. The measurement results are shown in Table 6 below.

[0153]

[0154] Experimental Example 2 (Drug Evaluation 1 Using Three-Dimensional Cellular Tissue) The effects of anticancer drugs on cancer cells were evaluated using the three-dimensional cellular tissues of Examples 1 and 2 and Comparative Examples 1 to 3 and the 2D culture of Comparative Example 4.

[0155] The three-dimensional cell tissues of Examples 1 and 2 and Comparative Examples 1 to 3 and the 2D culture of Comparative Example 4 were cultured for three days. The dedicated medium described above was used as the medium for Examples 1 and 2 and Comparative Examples 2 and 3. The general-purpose medium described above was used as the medium for Comparative Examples 1 and 4.

[0156] On day 4 of culture, naphthofluorescein (CAS No.: 61419-02-1), an anticancer drug, was added to the medium of each well of each three-dimensional cell tissue or 2D culture at a concentration of 0.01, 0.1, 1, or 10 μM. Tirapazamine (CAS No.: 27314-97-2), another anticancer drug, was added to the medium of each well of each three-dimensional cell tissue or 2D culture at a concentration of 0.01, 0.1, 1, or 10 μM.

[0157] Next, on the seventh day of culture, each three-dimensional cell tissue or 2D culture was treated with trypsin to dissociate the cells, and then the cancer cells were recovered by a pull-down assay using an anti-GFP antibody and stained with trypan blue. The number of cancer cells was then counted using an automated fluorescent cell counter (product name "Countess II FL", Thermo Fisher Scientific). GFP-positive and trypan blue-negative cells were determined to be viable cancer cells. Cell viability was calculated based on the following formula (1), and the effects of the anticancer drug on the cancer cells were evaluated according to the following evaluation criteria. The evaluation results are shown in Tables 7 and 8 below. Cell viability = number of surviving cancer cells in the presence of each concentration of anticancer drug / number of surviving cancer cells in the absence of anticancer drug × 100 (%) (1) (Evaluation criteria) I: Cell viability ≥ 90% II: 90% > cell viability ≥ 80% III: 80% > cell viability ≥ 70% IV: 70% > cell viability ≥ 50% V: cell viability ≤ 50%

[0158]

[0159]

[0160] In addition, the expression of hypoxia-related marker genes expressed by GFP-AsPC-1 in each three-dimensional cell tissue or 2D culture on day 7 of culture was measured by quantitative real-time PCR. APBA3 (MINT3) was measured as the hypoxia-related marker gene. Specifically, GFP-AsPC-1 in each three-dimensional cell tissue or 2D culture on day 7 of culture was recovered by pull-down assay using an anti-GFP antibody, and the expression level of the hypoxia-related marker gene was measured by quantitative real-time PCR. The measurement results are shown in Table 9 below. The values ​​in Table 9 are relative values, with the measured value in the 2D culture of Comparative Example 4 set as 1.

[0161]

[0162] As a result, it was revealed that the expression of hypoxia-related markers by cancer cells was significantly higher in the three-dimensional cellular tissue of the Example compared to the three-dimensional cellular tissue or 2D culture of the Comparative Example.Furthermore, it was confirmed that the effect of anticancer drugs on the reduction of cancer cells could be clearly confirmed in the three-dimensional cellular tissue of the Example compared to the three-dimensional cellular tissue or 2D culture of the Comparative Example.

[0163] [Example 3] (Production of three-dimensional cell tissue) GFP-AsPC-1 was produced by forcibly expressing the Mint3 gene, a molecule involved in the malignant transformation of cancer and inflammatory diseases. ＴＭ Using Transfection Reagent (Promega), Mint3 Human Tagged ORF Clone (Origene) was transfected into GFP-AsPC-1, forcing the Mint3 gene to be transiently expressed in GFP-AsPC-1.

[0164] Then, 1 × 10 ６ A three-dimensional cell tissue of Example 3 was obtained by producing a three-dimensional cell tissue in the same manner as in Example 2, except that GFP-AsPC-1 was used.

[0165] Comparative Example 5 (Production of 2D culture) 2 × 10 ６ The 2D culture of Comparative Example 5 was obtained in the same manner as in Comparative Example 4, except that GFP-AsPC-1 was used.

[0166] Experimental Example 3 (Drug Evaluation 2 Using Three-Dimensional Cellular Tissue) Using the three-dimensional cellular tissue of Example 3 and the 2D culture of Comparative Example 5, the effect of an anticancer drug on cancer cells was evaluated.

[0167] The three-dimensional cell tissue of Example 3 and the 2D culture of Comparative Example 5 were cultured for three days. The medium used in Example 3 was the dedicated medium described above. The medium used in Comparative Example 5 was the general-purpose medium described above.

[0168] On day 4 of culture, the anticancer drug naphthofluorescein was added to the medium of each well of each three-dimensional cell tissue or 2D culture at a concentration of 0.1, 1, or 10 μM. Subsequently, on day 7 of culture, each three-dimensional cell tissue or 2D culture was treated with trypsin to dissociate the cells, followed by staining with trypan blue. The number of viable cancer cells was then counted using an automated fluorescent cell counter (product name "Countess II FL," Thermo Fisher Scientific). GFP-positive and trypan blue-negative cells were determined to be viable cancer cells. Cell viability was calculated as in Experimental Example 2. The effects of the anticancer drug on cancer cells were evaluated according to the same evaluation criteria as in Experimental Example 2.

[0169] The evaluation results are shown in Table 10. For comparison, Table 10 also shows the results of Comparative Examples 1 to 4 and Examples 1 and 2 measured in Experimental Example 2.

[0170]

[0171] As a result, a comparison of the results of Comparative Examples 4 and 5 showed that in 2D cultures, expression of the Mint3 gene in cancer cells did not change the response to anticancer drugs. In contrast, a comparison of the results of Examples 2 and 3 showed that expression of the Mint3 gene in cancer cells resulted in a more pronounced effect of anticancer drugs on cancer cell reduction.

[0172] [Comparative Example 6] (Production of three-dimensional cell tissue) 1 × 10 ３ A three-dimensional cell tissue of Comparative Example 6 was obtained by producing a three-dimensional cell tissue in the same manner as in Comparative Example 1, except that RFP-HT29 was used.

[0173] [Comparative Example 7] (Production of three-dimensional cell tissue) 5 × 10 cells were used as the target cells. ３ A three-dimensional cell tissue of Comparative Example 7 was obtained by producing a three-dimensional cell tissue in the same manner as in Comparative Example 2, except that RFP-HT29 was used.

[0174] [Comparative Example 8] (Production of 2D culture) 1.0 × 10 ３ RFP-HT29 cells were suspended in 50 μL of general-purpose medium and seeded onto a 24-well plate to obtain the 2D culture of Comparative Example 8.

[0175] [Example 4] (Production of three-dimensional cellular tissue) In the production of the target cell compartment, 1 × 10 ５ A three-dimensional cell tissue of Example 4 was obtained by producing a three-dimensional cell tissue in the same manner as in Example 2, except that RFP-HT29 was used.

[0176] [Comparative Example 9] (Production of three-dimensional cell tissue) 1 × 10 cells were used as the target cells. ４ A three-dimensional cell tissue of Comparative Example 9 was obtained by producing a three-dimensional cell tissue in the same manner as in Comparative Example 1, except that GFP-A549 was used.

[0177] [Comparative Example 10] (Production of three-dimensional cell tissue) 1 × 10 cells were used as the target cells. ５ A three-dimensional cell tissue of Comparative Example 10 was obtained by producing a three-dimensional cell tissue in the same manner as in Comparative Example 2, except that GFP-A549 was used.

[0178] [Comparative Example 11] (Production of 2D culture) 1.0 × 10 ４ GFP-A549 cells were suspended in 50 μL of general-purpose medium and seeded onto a 24-well plate to obtain the 2D culture of Comparative Example 11.

[0179] [Example 5] (Production of three-dimensional cellular tissue) In the production of the target cell compartment, 1 × 10 ６ A three-dimensional cell tissue of Example 5 was obtained by producing a three-dimensional cell tissue in the same manner as in Example 2, except that GFP-A549 was used.

[0180] Experimental Example 4 (Analysis of Three-Dimensional Cellular Tissue) The three-dimensional cellular tissues of Examples 4 and 5 were cultured using the dedicated medium described above. Subsequently, on the third day after the start of culture, the cells were observed under a microscope, and the number of RFP-HT29 and GFP-A549 cells was counted based on the fluorescence of RFP or GFP. Furthermore, the percentage of the total volume of the target cells (RFP-HT29, GFP-A549) in the volume of the target cell compartment was measured. More specifically, fibrin, RFP regions, and GFP regions were stained by immunohistochemical staining (IHC). Subsequently, cross-sectional analysis was performed at five points per sample, and the volume of the RFP- or GFP-stained region relative to the volume of the fibrin-stained region was calculated, and the percentage of the total volume of the target cells in the volume of the target cell compartment was determined. The measurement results are shown in Table 11 below.

[0181]

[0182] Experimental Example 5 (Drug Evaluation 3 Using Three-Dimensional Cellular Tissues) The effects of hypoxic environment targeted drugs on cancer cells were evaluated using the three-dimensional cellular tissues of Examples 4 and 5, Comparative Examples 6, 7, 9, and 10, and the 2D cultures of Comparative Examples 8 and 11. The hypoxic environment targeted drug used was the HIF-1 inhibitor LW61 (CAS number: 934593-90-5).

[0183] The three-dimensional cell tissues of Examples 4 and 5, Comparative Examples 6, 7, 9, and 10, and the 2D cultures of Comparative Examples 8 and 11 were cultured for three days under normal oxygen conditions. The same 2D cultures as those of Comparative Examples 8 and 11 were also cultured for three days under hypoxic conditions with an oxygen concentration of 2% or less. A BIONIX hypoxic culture kit (Sugiyama Gen) was used for the culture under hypoxic conditions.

[0184] The above-mentioned dedicated medium was used as the medium for Examples 4 and 5 and Comparative Examples 6, 7, 9, and 10. The above-mentioned general-purpose medium was used as the medium for Comparative Examples 8 (normoxia), 8 (hypoxia), 11 (normoxia), and 11 (hypoxia).

[0185] On day 4 of culture, LW61 was added to the medium of each well of each three-dimensional cell tissue or 2D culture at 1, 10, or 100 μM. Subsequently, on day 7 of culture, each three-dimensional cell tissue or 2D culture was treated with trypsin to dissociate the cells, and cancer cells were recovered by pull-down assay using an anti-GFP antibody and stained with trypan blue. The number of cancer cells was then counted using an automated fluorescent cell counter (product name "Countess II FL," Thermo Fisher Scientific). GFP-positive and trypan blue-negative cells were determined to be live cancer cells.

[0186] The cell survival rate was calculated in the same manner as in Experimental Example 2. In addition, the effect of the drug on the cancer cells was evaluated according to the same evaluation criteria as in Experimental Example 2.

[0187] The evaluation results are shown in Tables 12 and 13 below.

[0188]

[0189]

[0190] As a result, it was shown that the effect of hypoxic environment targeted drugs in reducing cancer cells could be more significantly confirmed in the three-dimensional cell tissue of the example.

[0191] [Experimental Example 6] (Drug Evaluation 4 Using Three-Dimensional Cellular Tissue) The effects of anticancer drugs on cancer cells were evaluated using the three-dimensional cellular tissues of Example 1 and Comparative Example 1 and the 2D culture of Comparative Example 4. As anticancer drugs, a cytotoxic drug alone and a combination of a cytotoxic drug and an abnormal vascular targeting drug were investigated. Gemcitabine (CAS number: 95058-81-4) was used as the cytotoxic drug. Bevacizumab was used as the abnormal vascular targeting drug. The concentration of gemcitabine added was 10 μM. The concentration of bevacizumab added was 0.8 μg / mL.

[0192] The experimental procedure was the same as in Experimental Example 2, except for the different drugs. The drugs were evaluated using the following evaluation criteria. The experiment was performed five times to confirm reproducibility. The evaluation results are shown in Table 14 below. (Evaluation criteria) ◯: Compared to the survival rate of cancer cells when using a cytocidal drug alone, the survival rate of cancer cells when using a cytocidal drug in combination with an abnormal vascular targeting drug was significantly lower. ×: Compared to the survival rate of cancer cells when using a cytocidal drug alone, no significant change was observed in the survival rate of cancer cells when using a cytocidal drug in combination with an abnormal vascular targeting drug.

[0193]

[0194] As a result, it was shown that the three-dimensional cell tissue of the example can reproducibly confirm the combined effect of anticancer drugs compared to the three-dimensional cell tissue of the comparative example or 2D culture.

[0195] [Example 6] (Production of three-dimensional cell tissue) 1 x 10 ３ A three-dimensional cell tissue of Example 6 was obtained by producing a three-dimensional cell tissue in the same manner as in Example 2, except that RFP-HT29 was used.

[0196] [Example 7] (Production of three-dimensional cellular tissue) NHDF and HUVEC were prepared by forcibly expressing the MRC1 gene, a tumor-associated macrophage (TAM) / cancer-associated fibroblast (CAF) marker gene. ＴＭ Using Transfection Reagent (Promega), MRC1 Human Tagged ORF Clone (Origene) was transfected into a cell mixture of NHDF and HUVEC to transiently force-express the MRC1 gene.

[0197] A mixture of NHDF and HUVEC cells expressing the MRC1 gene was used as NHDF and HUVEC, and 1 × 10 ３ A three-dimensional cell tissue of Example 7 was obtained by producing a three-dimensional cell tissue in the same manner as in Example 2, except that RFP-HT29 was used.

[0198] [Comparative Example 12] (Production of three-dimensional cell tissue) 1 x 10 cancer cells ３A three-dimensional cell tissue of Comparative Example 12 was obtained by producing a three-dimensional cell tissue in the same manner as in Comparative Example 1, except that RFP-HT29 was used.

[0199] [Comparative Example 13] (Production of three-dimensional cell tissue) 1 x 10 cancer cells ３ A three-dimensional cell tissue of Comparative Example 13 was obtained by producing a three-dimensional cell tissue in the same manner as in Comparative Example 2, except that RFP-HT29 was used.

[0200] [Comparative Example 14] (Production of 2D culture) 1 x 10 ５ a mixture of NHDF and HUVEC cells not transfected with the MRC1 gene, and 1 × 10 ３ RFP-HT29 cells were suspended in 500 μL of general-purpose medium and seeded onto a 6-well plate to obtain the 2D culture of Comparative Example 14.

[0201] [Comparative Example 15] (Production of 2D culture) 1 x 10 ５ a mixture of NHDF and HUVEC cells expressing the MRC1 gene, and 1 x 10 ３ RFP-HT29 cells were suspended in 500 μL of general-purpose medium and seeded onto a 6-well plate to obtain the 2D culture of Comparative Example 15.

[0202] Experimental Example 7 (Evaluation of Drugs Using Three-Dimensional Cellular Tissues 5) The effects of drugs on cancer cells were evaluated using the three-dimensional cellular tissues of Examples 6 and 7 and Comparative Examples 12 and 13, and the 2D cultures of Comparative Examples 14 and 15. Anti-CD206 + docetaxel complex (Eurofins Genomics) was used as the drug. The amount of Anti-CD206 + docetaxel complex added was 0.01, 0.1, or 1 μM.

[0203] The three-dimensional cell tissues of Examples 6, 7 and Comparative Examples 12 and 13 and the 2D cultures of Comparative Examples 14 and 15 were cultured for three days. The medium used in Examples 6, 7 and Comparative Example 13 was the dedicated medium described above. The medium used in Comparative Examples 14 and 15 was the general-purpose medium described above.

[0204] On day 4 of culture, Anti-CD206+docetaxel Complex was added to the medium of each well of each three-dimensional cell tissue or 2D culture at concentrations of 0.01, 0.1, and 1 μM.

[0205] Next, on the seventh day of culture, each three-dimensional cell tissue or 2D culture was treated with trypsin to dissociate the cells, and then the cancer cells were recovered by a pull-down assay using an anti-RFP antibody and stained with trypan blue. The number of cancer cells was then counted using an automated fluorescent cell counter (product name "Countess II FL", Thermo Fisher Scientific). RFP-positive and trypan blue-negative cells were determined to be viable cancer cells. Cell viability was calculated as in Experimental Example 2. The effects of the drug on cancer cells were evaluated according to the same evaluation criteria as in Experimental Example 2. The evaluation results are shown in Table 15 below.

[0206]

[0207] As a result, it was shown that the drug-induced reduction of cancer cells could be confirmed at lower concentrations in the three-dimensional cell tissue of the examples.Furthermore, while no particular change in results was observed when TAM / CAF marker genes were expressed in stromal cells in 2D cultures, it was shown that the drug-induced reduction of cancer cells could be more significantly confirmed in the three-dimensional cell tissue of the examples by expressing TAM / CAF marker genes in stromal cells.

[0208] According to the present invention, it is possible to provide a three-dimensional cellular tissue in which the manifestation of medicinal effects can be clearly confirmed.

[0209] 1...three-dimensional cellular tissue, 11...interstitial cell compartment, 12...target cell compartment

Claims

1. A method for producing a three-dimensional cellular tissue, comprising: (a) obtaining a stromal cell-containing mixture comprising interstitial cells, a first cationic substance, a first extracellular matrix component, and a first polyelectrolyte; (b) gelling the interstitial cell-containing mixture to obtain a first gel composition comprising interstitial cells; (c) obtaining a target cell-containing mixture comprising target cells, a second cationic substance, a second extracellular matrix component, and a second polyelectrolyte; (d) placing the target cell-containing mixture in contact with the first gel composition comprising interstitial cells; (e) gelling the target cell-containing mixture to obtain a second gel composition comprising target cells; and (f) incubating the first gel composition comprising interstitial cells and the second gel composition comprising target cells to obtain a three-dimensional cellular tissue, wherein the proportion of the total volume of the target cells to the volume of the second gel composition comprising the target cells is 60 volume % or more.

2. The method of claim 1, further comprising the steps of: after step (e) and before step (f), placing a stromal cell-containing mixture containing the stromal cells, the first cationic substance, the first extracellular matrix component, and the first polyelectrolyte in contact with a second gel composition containing the target cells, allowing it to gel to form a third gel composition containing the stromal cells, and placing the second gel composition containing the target cells between the first and third gel compositions containing the stromal cells.

3. The manufacturing method described in claim 1 or 2, wherein the first gel composition containing the interstitial cells comprises a gel component in which at least one selected from the group consisting of extracellular matrix components, agarose, pectin and fibrin monomers has been gelled.

4. The manufacturing method described in claim 3, wherein the gel component is a fibrin gel formed by gelling fibrin monomers, and the step (b) of obtaining a first gel composition containing the interstitial cells includes a step of mixing thrombin and fibrinogen with the interstitial cell-containing mixture.

5. The method of claim 4, wherein the step (b) of obtaining a first gel composition containing the interstitial cells comprises the steps of: adding thrombin to the interstitial cell-containing mixture; and adding fibrinogen to the interstitial cell-containing mixture to which thrombin has been added, thereby forming a fibrin gel and gelling the interstitial cell-containing mixture.

6. The manufacturing method described in claim 1 or 2, wherein the second gel composition containing the target cells comprises a gel component in which at least one selected from the group consisting of extracellular matrix components, agarose, pectin and fibrin monomers has been gelled.

7. The manufacturing method described in claim 6, wherein the gel component is a fibrin gel formed by gelling fibrin monomers, and the step (e) of obtaining a second gel composition containing the target cells includes a step of mixing thrombin and fibrinogen with the target cell-containing mixture.

8. The manufacturing method described in claim 7, wherein step (e) of obtaining a second gel-like composition containing the target cells comprises the steps of: adding thrombin to the target cell-containing mixture; and adding fibrinogen to the target cell-containing mixture to which thrombin has been added, thereby forming a fibrin gel and gelling the target cell-containing mixture.

9. The method of claim 1 or 2, wherein the first and second extracellular matrix components are selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrin, proteoglycan, and combinations thereof.

10. The manufacturing method described in claim 1 or 2, wherein the target cells are cancer cells.

11. The manufacturing method described in claim 1 or 2, wherein the first cationic substance and the second cationic substance are the same, the first extracellular matrix component and the second extracellular matrix component are the same, and the first polymer electrolyte is the same as the second polymer electrolyte.

12. A three-dimensional cellular tissue, comprising an interstitial cell compartment containing interstitial cells, cationic substances, extracellular matrix components and polyelectrolytes, and a target cell compartment containing target cells, cationic substances, extracellular matrix components and polyelectrolytes, arranged in contact with the interstitial cell compartment containing interstitial cells, cationic substances, extracellular matrix components and polyelectrolytes, and the proportion of the total volume of the target cells to the volume of the target cell compartment is 60 volume % or more.

13. The three-dimensional cell tissue according to claim 12, wherein the target cells are cancer cells.

14. A method for evaluating a test substance, comprising the steps of: contacting a test substance with the three-dimensional cellular tissue according to claim 12 or 13; and evaluating the effect of the test substance on the target cells.

15. A method for detecting the effect of a test substance, comprising the steps of: contacting a test substance with the three-dimensional cellular tissue described in claim 12 or 13; and detecting the effect of the test substance on the target cells.

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