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

Abstract

This invention provides a technology to suppress the decrease in the thickness of three-dimensional cell tissue over time when three-dimensional cell tissue is created using mouse-derived cells. [Solution] A method for producing three-dimensional cell tissue, comprising the steps of: (A) obtaining a cell population containing mouse-derived stromal cells (excluding endothelial cells), a cationic substance, extracellular matrix components, and a polymer electrolyte; (B) obtaining a cell aggregate from the mixture; and (C) culturing the cell aggregate to obtain three-dimensional cell tissue, wherein the cell population further contains endothelial cells.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing a three-dimensional cell tissue and a three-dimensional cell tissue. This application claims priority to Japanese Patent Application No. 2020-140134 filed in Japan on August 21, 2020, the content of which is incorporated herein by reference.

Background Art

[0002] In recent years, in the fields of regenerative medicine and assay systems for drugs that require an environment close to the living body, the superiority of using three-dimensional cell tissues that are more three-dimensionally organized than cells grown on a flat plate has been shown. For this reason, various techniques for constructing three-dimensional cell tissues in vitro have been developed.

[0003] The inventors of the present application have previously developed a manufacturing technique for a three-dimensional cell tissue, which includes a step 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, a step of collecting the cells from the obtained mixture and forming a cell aggregate on a substrate, and a step of culturing the cells to obtain a three-dimensional cell tissue (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] For example, cancer cells and immune cells can be cultured inside or on the surface of a three-dimensional cell tissue to examine the immune cell response to cancer cells. In such cases, one way to eliminate the effects of rejection is to create a three-dimensional cell tissue using cells from the same strain of animal, for example, cells from the same strain of mouse, and then culture mouse-derived cancer cells and immune cells inside or on the surface of that three-dimensional cell tissue.

[0006] However, the inventors discovered that when three-dimensional cell tissues were created using mouse-derived cells, the thickness of the three-dimensional cell tissue decreased over time. For example, if the thickness of the three-dimensional cell tissue was 50 μm or more immediately after creation, it may become less than 10 μm after 4 days. This phenomenon is not observed when three-dimensional cell tissues are created using human-derived cells.

[0007] Therefore, the present invention aims to provide a technique for suppressing the decrease in the thickness of a three-dimensional cell tissue over time when a three-dimensional cell tissue is created using mouse-derived cells. [Means for solving the problem]

[0008] The present invention includes the following embodiments. [1] A method for producing a three-dimensional cell tissue, comprising the steps of (A) obtaining a cell population containing mouse-derived stromal cells (excluding endothelial cells), a cationic substance, an extracellular matrix component, and a polyelectrolyte; (B) obtaining a cell aggregate from the mixture; and (C) culturing the cell aggregate to obtain a three-dimensional cell tissue, wherein the cell population further contains mouse-derived endothelial cells and immune cells, the stromal cells, the endothelial cells, and the immune cells are syngenic, and the ratio of the number of endothelial cells to the number of stromal cells is 1.0% or more and 50% or less. [2] The manufacturing method according to [1], wherein the maximum thickness of a section obtained along a line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue on the fourth day after manufacturing is 50% or more of the maximum thickness of a section obtained along a line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue immediately after manufacturing. [3] The manufacturing method according to [1] or [2], wherein step (C) is performed after step (A) and step (B) are performed two or more times. [4] The extracellular matrix component is selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, enterin, fibrillin, proteoglycans and combinations thereof, as described in any of [1] to [3]. [5] The manufacturing method according to any one of [1] to [4], wherein the concentration of the extracellular matrix component in the mixture is 0.005 mg / mL or more and 1.0 mg / mL or less. [6] The polymer electrolyte is selected from the group consisting of glycosaminoglycans, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, and combinations thereof, as described in any one of [1] to [5]. [7] The manufacturing method according to any one of [1] to [6], wherein the concentration of the polymer electrolyte in the mixture is 0.005 mg / mL or more and 1.0 mg / mL or less. [8] The method for manufacturing according to any one of [1] to [7], wherein the interstitial cells are fibroblasts and the endothelial cells are vascular endothelial cells. [9] A manufacturing method according to any one of [1] to [8], wherein step (A) is performed in an aqueous medium.

[10] A three-dimensional cell tissue comprising mouse-derived stromal cells (excluding endothelial cells), a cell population including mouse-derived endothelial cells and immune cells, a cationic substance, extracellular matrix components and polyelectrolytes, wherein the ratio of the number of endothelial cells to the number of stromal cells in the cell population is 1.0% or more and 50% or less, and the stromal cells, endothelial cells and immune cells are syngenic.

[11] The three-dimensional cell tissue according to

[10] , wherein the maximum thickness of a section obtained along a line passing through the centroid when viewed from the upper surface of the three-dimensional cell tissue immediately after production is 40 μm or more.

[12] The three-dimensional cell tissue according to

[10] or

[11] , wherein the maximum thickness of a section obtained along a line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue on the fourth day after manufacture is 50% or more of the maximum thickness of a section obtained along a line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue immediately after manufacture. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a technique for suppressing the decrease in the thickness of a three-dimensional cell tissue over time when the three-dimensional cell tissue is prepared using mouse-derived cells. [Brief explanation of the drawing]

[0010] [Figure 1] These are micrographs of thin sections of three-dimensional cellular tissue taken in Experimental Example 1. [Figure 2] This graph shows the results of measuring the maximum thickness of the three-dimensional cell tissue, based on Figure 1. [Figure 3] This is a fluorescence micrograph showing the results of staining vascular endothelial cells in a three-dimensional cell tissue in Experimental Example 1. [Figure 4] These are micrographs of thin sections of three-dimensional cellular tissue taken in Experimental Example 2. [Modes for carrying out the invention]

[0011] [Method for producing three-dimensional cell tissue] In one embodiment, the present invention provides a method for producing a three-dimensional cell tissue, comprising the steps of (A) obtaining a cell population containing mouse-derived stromal cells (excluding endothelial cells), a cationic substance, an extracellular matrix component, and a polyelectrolyte; (B) obtaining a cell aggregate from the mixture; and (C) culturing the cell aggregate to obtain a three-dimensional cell tissue, wherein the cell population further contains endothelial cells.

[0012] The manufacturing method of this embodiment includes a step (A) of obtaining a mixture containing mouse-derived stromal cells (excluding endothelial cells) and endothelial cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte, a step (B) of obtaining a cell aggregate from the mixture, and a step (C) of culturing the cell aggregate to obtain a three-dimensional cell tissue. It can also be said that it is a manufacturing method of a three-dimensional cell tissue.

[0013] In this specification, "three-dimensional cell tissue" means a three-dimensional aggregate of cells. The three-dimensional cell tissue produced by the manufacturing method of this embodiment includes at least endothelial cells and mouse-derived stromal cells other than endothelial cells.

[0014] Examples of the uses of three-dimensional cell tissue include, but are not limited to, a living tissue model and a solid cancer model. Examples of the living tissue model include models of skin, hair, bone, cartilage, teeth, cornea, blood vessels, lymphatic vessels, heart, liver, pancreas, nerves, and esophagus. Examples of the solid cancer model include models of gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell cancer, and liver cancer.

[0015] For example, when analyzing the behavior of immune cells against cancer cells, immune cells can be further included in the three-dimensional cell tissue. In this case, it is preferable that all the cells constituting the three-dimensional cell tissue are syngeneic.

[0016] There is no particular limitation 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 cell culture insert, or a three-dimensional cell tissue formed by culturing cells in a scaffold composed of a natural biopolymer such as collagen or a synthetic polymer, or it may be a cell aggregate (spheroid), or it may be a sheet-like cell structure.

[0017] The inventors discovered that when producing three-dimensional cell tissue using mouse-derived cells, the decrease in thickness over time can be suppressed by including endothelial cells in the cell population used, thus completing the present invention.

[0018] According to the manufacturing method of this embodiment, even when a three-dimensional cell tissue is produced using mouse-derived cells, the decrease in the thickness of the three-dimensional cell tissue over time can be suppressed. As an example of the degree to which the decrease in the thickness of the three-dimensional cell tissue over time is suppressed, the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue on the fourth day after production is 50% or more of the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue immediately after production. Here, the maximum value of the three-dimensional cell tissue on the fourth day after production can also be said to be the maximum thickness of the three-dimensional cell tissue measured in a section obtained by cutting the three-dimensional cell tissue along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue four days after the start of culture. Furthermore, the maximum value of the three-dimensional cell tissue immediately after production can also be said to be the maximum thickness of the three-dimensional cell tissue measured in a section obtained by cutting the three-dimensional cell tissue along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue immediately after production. Here, "immediately after manufacturing" may refer to 5 minutes to 72 hours after the start of cell aggregate culture in step (C), or it may refer to 1 day (preferably 24 hours) after the start of cell aggregate culture in step (C). Also, "4th day after manufacturing" may refer to 4 days (preferably 96 hours) after the start of cell aggregate culture in step (C).

[0019] Here, the thickness of the section obtained along the line passing through the centroid when viewed from the top surface of the three-dimensional cell tissue is the thickness of the section at approximately the center of the three-dimensional cell tissue. The shape of the three-dimensional cell tissue varies depending on the container used to manufacture it. For example, if the three-dimensional cell tissue is manufactured using a cylindrical cell culture insert, it will be cylindrical. In this case, the shape of the three-dimensional cell tissue when viewed from the top surface is a circle, and the centroid when viewed from the top surface is the center of the circle. The shape of the three-dimensional cell tissue is not limited to a cylindrical shape and can be any shape depending on the purpose. Specifically, examples include polygonal prism shapes such as triangular prisms and rectangular prisms.

[0020] The manufacturing method of this embodiment includes the steps of (A) obtaining a mixture containing mouse-derived stromal cells (excluding endothelial cells), a cell population including endothelial cells, a cationic substance, extracellular matrix components, and a polyelectrolyte; (B) obtaining a cell aggregate from the mixture; and (C) culturing the cell aggregate to obtain a three-dimensional cell tissue. Each step will be described below.

[0021] First, in step (A), a mixture is obtained containing mouse-derived stromal cells (excluding endothelial cells), a cell population including endothelial cells, a cationic substance, extracellular matrix components, and a polymer electrolyte. Step (A) is preferably carried out in an aqueous medium.

[0022] Stromal cells are a general term for cells that make up the supporting tissue of epithelial cells. Examples of stromal cells include fibroblasts and smooth muscle cells. Whether or not a cell is a stromal cell can be determined by the morphology of the cell observed under a microscope, or by the expression of marker molecules in the cell.

[0023] Markers for fibroblasts include fibroblast growth factor receptor (FGFR)1, FGFR2, FGFR3, CD90, and vimentin. Markers for smooth muscle cells include actin, desmin, carbonin, and SM22.

[0024] In the manufacturing method of this embodiment, mouse-derived cells are used as stromal cells. One type of stromal cell may be used alone, or two or more types may be used in mixture. In addition to mouse-derived stromal cells, stromal cells derived from species other than mice may also be used. Examples of species other than mice include humans, monkeys, dogs, cats, rabbits, pigs, cattle, and rats.

[0025] Examples of endothelial cells include vascular endothelial cells and lymphatic endothelial cells, but vascular endothelial cells are preferred. The origin of the endothelial cells is not particularly limited and includes, for example, humans, monkeys, dogs, cats, rabbits, pigs, cattle, mice, and rats. Among these, mouse-derived endothelial cells are preferred.

[0026] Whether or not a cell is an endothelial cell can be determined by its morphology observed under a microscope, or by the expression of marker molecules within the cell.

[0027] Markers for vascular endothelial cells include CD31, VEGFR-2, and Tie-2 / Tek. Markers for lymphatic endothelial cells include podoplanin, LYVE-1, PROX-1, and VEGFR-3.

[0028] In the manufacturing method of this embodiment, it is preferable that the interstitial cells are fibroblasts and the endothelial cells are vascular endothelial cells.

[0029] In the manufacturing method of this embodiment, the ratio of endothelial cells to the number of stromal cells (excluding endothelial cells) in the cell population is preferably 1.0% to 50%, but may also be 1.0% to 20%, 1.5% to 20%, or 1.5% to 10%. As will be described later in the examples, when the proportion of endothelial cells is within the above range, it tends to suppress the decrease in the thickness of the three-dimensional cell tissue over time, even when a three-dimensional cell tissue is made using mouse-derived cells.

[0030] The cell population may include mouse-derived stromal cells (excluding endothelial cells) and cells other than endothelial cells. Examples of such cells include somatic cells derived from bone, muscle, internal organs, nerves, brain, skin, blood, etc., germ cells, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), tissue stem cells, and cancer cells. Examples of somatic cells derived from blood include immune cells such as lymphocytes, neutrophils, macrophages, and dendritic cells. Examples of cancer cells include gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, and liver cancer.

[0031] The cells that make up the cell population may be primary cells, or cultured cells such as subcultured cells or cell lines.

[0032] As cationic substances, any positively charged substance can be used, as long as it does not adversely affect cell growth and cell aggregate formation. 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.

[0033] The concentration of the cationic substance in step (A) is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. 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, 30 to 80 mM, 40 to 70 mM, or 45 to 60 mM.

[0034] 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 cell growth and cell aggregate formation. 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 7.4.

[0035] Any component constituting the extracellular matrix (ECM) can be used as the extracellular matrix component, as long as it does not adversely affect cell growth and cell aggregate formation. Examples of extracellular matrix components include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, enterin, fibrillin, proteoglycans, and their modified or variant forms. Extracellular matrix components may be used individually or in combination of two or more.

[0036] Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan. Among the extracellular matrix components, collagen, laminin, and fibronectin are preferred, with collagen being particularly preferred.

[0037] The concentration of the extracellular matrix component is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation, but is preferably greater than 0 mg / mL and less than 1.0 mg / mL. The concentration of the extracellular matrix component may be 0.005 mg / mL or more and 1.0 mg / mL or less, 0.01 mg / mL or more and 1.0 mg / mL or less, 0.025 mg / mL or more and 1.0 mg / mL or less, or 0.025 mg / mL or more and 0.1 mg / mL or less. The extracellular matrix component can be used dissolved in a suitable solvent. Examples of solvents include, but are not limited to, water, buffer solutions, and aqueous acetic acid solutions. Among these, buffer solutions or aqueous acetic acid solutions are preferred. The pH of the buffer solution and aqueous acetic acid solution is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation.

[0038] In this specification, a polyelectrolyte means a polymer having dissociable functional groups in its polymer chain. Any polyelectrolyte can be used as the polyelectrolyte in this embodiment, as long as it does not adversely affect cell growth and cell aggregate formation. Examples of polyelectrolytes include, but are not limited to, heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate and chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, and glycosaminoglycans such as hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, and derivatives thereof. These polyelectrolytes may be used individually or in combination of two or more.

[0039] The polymer electrolyte used in this embodiment is preferably a glycosaminoglycan. Among these, heparin, chondroitin sulfate, and dermatan sulfate are preferred, and heparin is particularly preferred.

[0040] The concentration of the polyelectrolyte in the manufacturing method of this embodiment is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. The concentration of the polyelectrolyte is preferably greater than 0 mg / mL and less than 1.0 mg / mL, may be 0.005 mg / mL or more and 1.0 mg / mL or less, may be 0.01 mg / mL or more and 1.0 mg / mL or less, may be 0.025 mg / mL or more and 1.0 mg / mL or less, or may be 0.025 mg / mL or more and 0.1 mg / mL or less.

[0041] The polymer electrolyte can be used after being dissolved in a suitable solvent. Examples of solvents include, but are not limited to, water and buffer solutions. When a cationic buffer solution is used as the cationic substance mentioned above, the polymer electrolyte may also be used after being dissolved in the cationic buffer solution.

[0042] In the manufacturing method of this embodiment, the mixing ratio (final concentration ratio) of the polymer electrolyte and the extracellular matrix component is preferably 1:2 to 2:1, but may also be 1:1.5 to 1.5:1, or 1:1.

[0043] In step (A), the mixing of mouse-derived stromal cells (excluding endothelial cells), cell populations containing endothelial cells, cationic substances, extracellular matrix components, and polymer electrolytes can be carried out in a suitable container such as a dish, tube, flask, bottle, or plate. This mixing may also be carried out in the container used in step (B).

[0044] Next, in step (B), a cell aggregate is obtained from the mixture obtained in step (A). In this specification, "cell aggregate" means a structure in which cells have assembled together into a single unit. The cell aggregate also includes cell precipitates obtained by centrifugation or filtration. In one embodiment, the cell aggregate is a slurry-like viscous substance. "Slurry-like viscous substance" refers to a gel-like cell aggregate as 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.

[0045] Cell aggregates can be formed by placing the mixture obtained in step (A) into a suitable container and allowing it to stand, or by placing the mixture obtained in step (A) into a suitable container and collecting the cells by, for example, centrifugation, magnetic separation, or filtration to form cell aggregates. When cells are collected by centrifugation, magnetic separation, or filtration, the liquid portion may or may not be removed.

[0046] Examples of containers used in process (B) include culture vessels for culturing cells. Culture vessels may be containers made of materials and shaped in a manner commonly used for culturing cells and microorganisms. Examples of culture vessel materials include, but are not limited to, glass, stainless steel, and plastic. Examples of culture vessels include, but are not limited to, dishes, tubes, flasks, bottles, and plates. Preferably, at least a portion of the container is made of a material that allows liquid to pass through but does not allow cells in the liquid to pass through. Examples of such containers include, but are not limited to, cell culture inserts such as Transwell® inserts, Netwell® inserts, Falcon® cell culture inserts, and Millicell® cell culture inserts.

[0047] The conditions for centrifugation are not particularly limited, as long as they do not adversely affect cell growth. For example, cells can be collected by seeding the mixture into a cell culture insert and centrifuging it at 10°C and 400×g for 1 minute.

[0048] Next, in step (C), the cell aggregate obtained in step (B) is cultured to obtain a three-dimensional cell tissue. The cell culture in step (C) 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 according to the type of cells and the desired function. The culture medium is not particularly limited, but examples include D-MEM, E-MEM, MEMα, RPMI-1640, McCoy's 5A, Ham's F-12, etc., and media to which serum is added in an amount of about 1-20% by volume. Examples of serum include bovine serum (CS), fetal bovine serum (FBS), and fetal equine serum (HBS). Various conditions of the culture environment, such as temperature and atmospheric composition, should also be adjusted to conditions suitable for the cells to be cultured.

[0049] Next, in step (C), the cell aggregate obtained in step (B) is cultured to obtain a three-dimensional cell tissue. The time for culturing the cell aggregate to obtain the three-dimensional cell tissue may be 5 minutes to 168 hours, 12 hours to 144 hours, or 24 hours to 72 hours. Step (C) has the effect of promoting cell adhesion within the cell aggregate, resulting in a stable three-dimensional cell tissue.

[0050] The cell aggregates may be suspended in a solution before culturing. The solution is not particularly limited, as long as it does not adversely affect cell growth and the formation of three-dimensional cell tissue. For example, a culture medium or buffer suitable for the cells constituting the cell aggregates can be used. The suspension of the cell aggregates can be done in a suitable container such as a dish, tube, flask, bottle, or plate.

[0051] When cell aggregates are suspended in a solution, the cells may be precipitated to form a cell precipitate before culturing. Cell precipitation can be performed, for example, by centrifugation. The centrifugation conditions are not particularly limited, as long as they do not adversely affect cell growth and cell aggregate formation. For example, the cell aggregate suspension may be precipitated by centrifugation at room temperature at 400-1,000 × g for 1 minute. Alternatively, the cells may be precipitated by natural sedimentation.

[0052] The container used in process (C) is the same as the container used in process (B). In process (C), the container used in process (B) may be used as is, or the contents may be transferred to a different container.

[0053] During cell culture, substances may be added to the culture medium to suppress deformation of the constructed three-dimensional cell tissue (e.g., tissue contraction, detachment of tissue ends, etc.). Examples of such substances include, but are not limited to, Y-27632, a Rho-associated coiled-coil forming kinase / Rho-binding kinase (ROCK) inhibitor.

[0054] Steps (A) and (B) may be performed two or more times before step (C). By repeating steps (A) and (B), cell aggregates or cell precipitates can be stacked to produce a three-dimensional cell tissue having multiple layers. In other words, a three-dimensional cell tissue with a large thickness can be produced.

[0055] Furthermore, when repeating steps (A) and (B) to layer cell aggregates or cell precipitates, a different cell population may be used each time to layer three-dimensional cell tissues composed of different types of cells. For example, after performing steps (A) and (B) for the first time, a second step (A) may be performed using a different cell population than that used in the first step (A). Then, by performing the second step (B), a layer containing the cell population used in the second step (A) can be formed on top of a layer containing the cell population used in the first step (A). By repeating steps (A) and (B) multiple times in this way, three-dimensional cell tissues composed of multiple types of cell populations can be layered.

[0056] [3D cell tissue] In one embodiment, the present invention provides a three-dimensional cell tissue comprising mouse-derived stromal cells (excluding endothelial cells) and a cell population including endothelial cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte, wherein the ratio of the number of endothelial cells to the number of stromal cells (excluding endothelial cells) in the cell population is 1.0% or more and 50% or less.

[0057] In this embodiment, it is preferable that the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from above is 40 μm or more. In other words, it is preferable that the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from above is 40 μm or more immediately after manufacturing the three-dimensional cell tissue. The maximum value of the three-dimensional cell tissue immediately after manufacturing can be rephrased as described above.

[0058] Despite being formed from mouse-derived cells, the three-dimensional cell tissue of this embodiment exhibits suppressed reduction in thickness over time. An example of the degree to which the reduction in thickness of the three-dimensional cell tissue over time is suppressed is that the maximum thickness of a section obtained along a line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue four days after manufacturing is 50% or more of the maximum thickness of a section obtained along a line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue immediately after manufacturing. The aforementioned maximum value of the three-dimensional cell tissue four days after manufacturing can be rephrased as described above.

[0059] In the three-dimensional cell tissue of this embodiment, the stromal cells, endothelial cells, cationic substances, extracellular matrix components, and polyelectrolytes are the same as those described above.

[0060] In the three-dimensional cell tissue of this embodiment, the ratio of endothelial cells to the total number of stromal cells (excluding endothelial cells) in the cell population is 1.0% or more and 50% or less, may be 1.0% or more and 20% or less, or 1.0% or more and 10% or less. [Examples]

[0061] 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.

[0062] [Experimental Example 1] (Manufacturing of three-dimensional cell tissue 1) Three-dimensional cell tissues were fabricated. Mouse embryonic fibroblasts (MEF, Sciencell) were used as stromal cells. Mouse colon-derived vascular endothelial cells (Cell Biologics) were used as endothelial cells.

[0063] MEF and the aforementioned mouse colon-derived vascular endothelial cells were suspended in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1 mg / mL heparin and 0.1 mg / mL collagen in various proportions to prepare cell suspensions with endothelial cell-to-stromal cell ratios of 0%, 1.5%, 3%, 4.5%, 10%, and 20%. Collagen I was used as the collagen.

[0064] Next, each cell suspension was centrifuged at 4°C and 400 × g for 3 minutes, the supernatant was removed, and then each was resuspended in an appropriate amount of DMEM medium containing 10% fetal bovine serum (FBS). Subsequently, each cell suspension was placed in a 24-well cell culture insert with an MEF of 1.5 × 10⁶ per well. 6 The seeds were sown so that each seed was individually distributed. The bottom surface area per well of the cell culture insert was 33 mm². 2 That was the case.

[0065] Next, the cell culture insert was centrifuged at 4°C and 400 × g (gravitational acceleration) for 1 minute to obtain cell aggregates. Subsequently, an appropriate amount of culture medium was added to the cell culture insert, and the cells were cultured in a CO2 incubator (37°C, 5% CO2) for 1 week. The culture medium was changed as needed during this period.

[0066] One day after the start of culture of the cell aggregates (24 hours later, i.e., immediately after production) and four days later (96 hours later, i.e., four days after production), each three-dimensional cell tissue was fixed using 10% Mildform (Fujifilm Wako Pure Chemical Industries). Subsequently, each three-dimensional cell tissue was embedded in paraffin, and thin sections were prepared along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue. The thin sections were then stained with hematoxylin-eosin (HE) and observed under a microscope to measure the maximum thickness of the three-dimensional cell tissue.

[0067] Figure 1 shows micrographs of thin sections of each three-dimensional cell tissue. The scale bar is 100 μm. In Figure 1, the percentage (%) indicates the ratio of endothelial cells to stromal cells used in the production of the three-dimensional cell tissue. "Immediately after production" and "4 days after production" indicate the three-dimensional cell tissue fixed immediately after production and 4 days after production, respectively.

[0068] Figure 2 is a graph showing the results of measuring the maximum thickness of each three-dimensional cell tissue based on Figure 1. In Figure 2, the vertical axis represents the maximum thickness of the three-dimensional cell tissue, and the horizontal axis represents the time (days) since the start of culture of the cell aggregate. "Colon EC" refers to mouse colon-derived vascular endothelial cells, and the percentage (%) represents the ratio of endothelial cells to the number of stromal cells used to produce the three-dimensional cell tissue. Table 1 shows the results of measuring the maximum thickness of each three-dimensional cell tissue immediately after production and four days after production.

[0069] [Table 1]

[0070] The results revealed that the thickness of the three-dimensional cell tissue without endothelial cells significantly decreased on the fourth day after the start of cell aggregate culture. In contrast, the decrease in thickness on the fourth day after the start of cell aggregate culture was significantly suppressed in the three-dimensional cell tissue containing endothelial cells. Specifically, the maximum thickness of the section obtained along the line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue on the fourth day after production was more than 50% of the maximum thickness of the section obtained along the line passing through the centroid as viewed from the top surface of the three-dimensional cell tissue immediately after production.

[0071] Furthermore, vascular endothelial cells in three-dimensional cell tissue were stained with anti-CD31 antibody and observed. Figure 3 shows a photograph of the three-dimensional cell tissue after staining with anti-mouse CD31 rat antibody (clone MEC13.3, BD Biosciences) as the primary antibody, followed by staining with Alexafluoro 488-labeled anti-rat IgG antibody (Thermo Fisher Scientific) as the secondary antibody, and then observing the results with a fluorescence microscope. The scale bar is 2 mm. As mentioned above, the bottom surface area per well of the culture insert is 33 mm². 2 That was the case.

[0072] In Figure 3, "colon EC" refers to mouse colon-derived vascular endothelial cells, and the percentage (%) indicates the ratio of endothelial cells to the number of stromal cells used in the production of the three-dimensional cell tissue. Furthermore, "Immediately after production" and "4 days after production" indicate the results for the three-dimensional cell tissue immediately after production and 4 days after production, respectively.

[0073] [Experimental Example 2] (Manufacturing of three-dimensional cell tissue 2) The three-dimensional cell tissue was prepared in the same manner as in Experimental Example 1, except that only stromal cells were used as the cells. Normal human dermal fibroblasts (NHDF) were used as the stromal cells. Specifically, NHDF was suspended in 50 mM Tris-HCl buffer (pH 7.4) containing 0.1 mg / mL heparin and 0.1 mg / mL collagen. Collagen I was used as the collagen.

[0074] Next, the cell suspension was centrifuged at 4°C and 400×g for 3 minutes, the supernatant was removed, and the cell suspension was resuspended in an appropriate amount of DMEM medium containing 10% fetal bovine serum (FBS). Subsequently, the cell suspension was placed in 24-well cell culture inserts, 2.0×10⁶ cells per well. 6 The seeds were sown individually. The bottom surface area per well of the cell culture insert was 33 mm². 2 That was the case.

[0075] Next, the cell culture insert was centrifuged at 4°C and 400 × g (gravitational acceleration) for 1 minute to obtain cell aggregates. Subsequently, an appropriate amount of culture medium was added to the cell culture insert, and the cells were cultured in a CO2 incubator (37°C, 5% CO2) for 1 week. The culture medium was changed as needed during this period.

[0076] One day after the start of culture of the cell aggregates (24 hours later, i.e., immediately after production) and five days later (120 hours later, i.e., 5 days after production), each three-dimensional cell tissue was fixed using 10% Mildform (Fujifilm Wako Pure Chemical Industries). Subsequently, each three-dimensional cell tissue was embedded in paraffin, and thin sections were prepared along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue. Then, the thin sections were stained with hematoxylin-eosin (HE) and observed under a microscope to measure the maximum thickness of the three-dimensional cell tissue.

[0077] Figure 4 shows micrographs of thin sections of three-dimensional cell tissue. The scale bar is 100 μm. In Figure 4, "Immediately after production" and "5 days after production" indicate three-dimensional cell tissue fixed immediately after production and 5 days after production, respectively.

[0078] As a result, the maximum thickness of the three-dimensional cell tissue immediately after the production of the cell aggregate was 85.8 μm. Furthermore, the maximum thickness of the three-dimensional cell tissue 5 days after the production of the cell aggregate was 54.2 μm. From these results, it was clear that when three-dimensional cell tissue was created using human-derived cells, the decrease in thickness over time was significantly less compared to when three-dimensional cell tissue was created using mouse-derived cells. [Industrial applicability]

[0079] According to the present invention, it is possible to provide a technique for suppressing the decrease in the thickness of a three-dimensional cell tissue over time when the three-dimensional cell tissue is prepared using mouse-derived cells.

Claims

1. Step (A) to obtain a mixture containing a cell population including mouse-derived stromal cells (excluding endothelial cells), a cationic substance, extracellular matrix components, and a polymer electrolyte, The process of obtaining a cell aggregate from the mixture (B), The process includes (C) culturing the aforementioned cell aggregate to obtain a three-dimensional cell tissue, The aforementioned cell population further includes mouse-derived endothelial cells and immune cells. The stromal cells, endothelial cells, and immune cells are syngenic, A method for producing a three-dimensional cell tissue, wherein the ratio of the number of endothelial cells to the number of stromal cells is 1.0% or more and 50% or less.

2. The manufacturing method according to claim 1, wherein the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue on the fourth day after manufacturing is 50% or more of the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue immediately after manufacturing.

3. The manufacturing method according to claim 1 or 2, wherein step (C) is performed after step (A) and step (B) are performed two or more times.

4. The method for producing the extracellular matrix according to any one of claims 1 to 3, wherein the extracellular matrix component is selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, enterin, fibrillin, proteoglycans, and combinations thereof.

5. The manufacturing method according to any one of claims 1 to 4, wherein the concentration of the extracellular matrix component in the mixture is 0.005 mg / mL or more and 1.0 mg / mL or less.

6. The method for producing the polymer electrolyte according to any one of claims 1 to 5, wherein the polymer electrolyte is selected from the group consisting of glycosaminoglycans, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, and combinations thereof.

7. The manufacturing method according to any one of claims 1 to 6, wherein the concentration of the polymer electrolyte in the mixture is 0.005 mg / mL or more and 1.0 mg / mL or less.

8. The manufacturing method according to any one of claims 1 to 7, wherein the interstitial cells are fibroblasts and the endothelial cells are vascular endothelial cells.

9. The manufacturing method according to any one of claims 1 to 8, wherein step (A) is carried out in an aqueous medium.

10. It contains mouse-derived stromal cells (excluding endothelial cells), a cell population including mouse-derived endothelial cells and immune cells, a cationic substance, extracellular matrix components, and polyelectrolytes. The ratio of the number of endothelial cells to the number of stromal cells in the cell population is 1.0% or more and 50% or less. A three-dimensional cell tissue in which the stromal cells, endothelial cells, and immune cells are syngenic.

11. The three-dimensional cell tissue according to claim 10, wherein the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from the upper surface of the three-dimensional cell tissue immediately after manufacturing is 40 μm or more.

12. The three-dimensional cell tissue according to claim 10 or 11, wherein the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue on the fourth day after manufacture is 50% or more of the maximum thickness of a section obtained along a line passing through the center of gravity when viewed from the top surface of the three-dimensional cell tissue immediately after manufacture.

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