Method for manufacturing a three-dimensional tissue and method for promoting differentiation of adipose-derived stem cells

Abstract

To provide a method for promoting the differentiation of adipose-derived stem cells into mature adipocytes in the production of a three-dimensional tissue containing mature adipocytes. [Solution] A method for producing a three-dimensional tissue body containing mature adipocytes, comprising incubating cells containing at least adipose-derived stem cells in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a three-dimensional tissue and a method for promoting the differentiation of adipose-derived stem cells. [Background technology]

[0002] As methods for artificially creating structures that mimic biological tissue, for example, a method for producing a three-dimensional tissue (Patent Document 1) that includes arranging cells coated with a collagen-containing film in a three-dimensional manner to form a three-dimensional tissue, and a method for producing a three-dimensional cell tissue (Patent Document 2) that includes mixing cells with a cationic substance and extracellular matrix components to obtain a mixture, collecting cells from the obtained mixture, and forming a cell aggregate on a substrate. Furthermore, the present inventors have proposed a method (Patent Document 3) for producing a large three-dimensional tissue with a thickness of 1 mm or more using a relatively small number of cells by bringing cells into contact with fragmented exogenous collagen. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2015 / 072164 [Patent Document 2] International Publication No. 2017 / 146124 [Patent Document 3] International Publication No. 2018 / 143286 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] According to the manufacturing method described above, a three-dimensional tissue, which is an aggregate of cells artificially created by cell culture, can be obtained. In particular, three-dimensional tissues containing adipocytes are expected to be used as substitutes for experimental animals, transplant materials, etc., and a method to promote the differentiation of adipose-derived stem cells when manufacturing three-dimensional tissues containing adipocytes is desired.

[0005] This invention has been made in view of the above circumstances, and aims to provide a method for promoting the differentiation of adipose-derived stem cells into mature adipocytes in the production of a three-dimensional tissue containing mature adipocytes. [Means for solving the problem]

[0006] As a result of diligent research, the inventors discovered that incubation of adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor and / or specific fatty acids promotes the differentiation of adipose-derived stem cells into mature adipocytes, thus completing the present invention.

[0007] In other words, the present invention includes, for example, the following inventions. [1] A method for producing a three-dimensional tissue containing mature adipocytes, comprising incubating cells containing at least adipose-derived stem cells in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. [2] The method for producing the cells described above, comprising incubating the cells in the presence of a TGFβ type I receptor inhibitor, as described in [1]. [3] The method for producing the product according to [2], wherein incubation in the presence of the TGFβ type I receptor inhibitor is incubation in a culture medium containing the TGFβ type I receptor inhibitor, and the content of the TGFβ type I receptor inhibitor in the culture medium is 1 μM or more and 10 μM or less. [4] The method according to any one of [1] to [3], wherein the cells containing at least adipose-derived stem cells do not contain mature adipocytes. [5] The manufacturing method according to any one of items [1] to [4], wherein the above-mentioned adipose-derived stem cells are derived from bovine tissue. [6] The manufacturing method according to any one of items [1] to [5], wherein incubation is performed for 96 hours or more and 384 hours or less. [7] The production method according to any one of [1] to [6], comprising incubating the above cells together with fragmented extracellular matrix components in the presence of a TGFβ type I receptor inhibitor. [8] The production method according to [7], wherein in the above three-dimensional tissue construct, the fragmented extracellular matrix components are arranged in the gaps between the above cells. [9] The production method according to [7] or [8], wherein the fragmented extracellular matrix components are fragmented collagen components.

[10] The production method according to any one of [7] to [9], further comprising a step of contacting the above cells with the fragmented extracellular matrix components in an aqueous medium before incubation.

[11] A method for promoting the differentiation of adipose-derived stem cells, comprising incubating cells containing at least adipose-derived stem cells in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid.

[12] The method according to

[11] , comprising incubating the above cells in the presence of a TGFβ type I receptor inhibitor.

[13] Incubating in the presence of the above TGFβ type I receptor inhibitor is incubating in a medium containing the TGFβ type I receptor inhibitor, and the content of the TGFβ type I receptor inhibitor in the medium is 1 μM or more and 10 μM or less. The method according to

[12] .

[14] The method according to any one of

[11] to

[13] , wherein the adipose-derived stem cells are of bovine origin.

[15] The method according to any one of

[11] to

[14] , wherein the incubation is carried out for 96 hours or more and 384 hours or less.

[16] The method according to any one of

[11] to

[15] , comprising incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of a TGFβ type I receptor inhibitor.

[17] The method according to

[16] , wherein the above-mentioned fragmented extracellular matrix component is a fragmented collagen component.

[18] The method according to

[16] or

[17] , further comprising the step of contacting the cells with the fragmented extracellular matrix components in an aqueous medium before incubation. [P1] A method for producing a three-dimensional tissue containing mature adipocytes, comprising incubating cells containing at least adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor. [P2] The method for producing the above cells according to [P1], comprising incubating the above cells in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. [P3] The method according to [P1] or [P2], wherein the cells containing at least adipose-derived stem cells do not contain mature adipocytes. [P4] The method for producing the product according to any one of [P1] to [P3], wherein incubation in the presence of the TGFβ type I receptor inhibitor is performed in a culture medium containing the TGFβ type I receptor inhibitor, and the content of the TGFβ type I receptor inhibitor in the culture medium is 1 μM or more and 10 μM or less. [P5] The manufacturing method described in any of [P1] to [P4], wherein the above-mentioned adipose-derived stem cells are derived from bovine tissue. [P6] A manufacturing method according to any one of [P1] to [P5], wherein incubation is performed for 96 hours or more and 384 hours or less. [P7] A method for producing the above cells according to any one of [P1] to [P6], comprising incubating the above cells together with fragmented extracellular matrix components in the presence of a TGFβ type I receptor inhibitor. [P8] The manufacturing method described in [P7], wherein the fragmented extracellular matrix components are arranged in the spaces between the cells in the three-dimensional tissue described above. [P9] The method for producing the product according to [P7] or [P8], wherein the above-mentioned fragmented extracellular matrix component is a fragmented collagen component. [P10] The method for producing the cells described above and the fragmented extracellular matrix components described above in any one of [P7] to [P9], further comprising the step of contacting the cells described above with the fragmented extracellular matrix components in an aqueous medium before incubation. [P11] A method for promoting the differentiation of adipose-derived stem cells, comprising incubating cells containing at least adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor. [P12] The method according to [P11], wherein incubation in the presence of the TGFβ type I receptor inhibitor is incubation in a culture medium containing the TGFβ type I receptor inhibitor, and the content of the TGFβ type I receptor inhibitor in the culture medium is 1 μM or more and 10 μM or less. [P13] The method according to [P11] or [P12], wherein the adipose-derived stem cells described above are derived from bovine tissue. [P14] The incubation is performed for 96 hours or more and 384 hours or less, using one of the methods described in [P11] to [P13]. [P15] A method according to any one of [P11] to [P14], comprising incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of a TGFβ type I receptor inhibitor. [P16] The method according to [P15], wherein the above-mentioned fragmented extracellular matrix component is a fragmented collagen component. [P17] The method according to [P15] or [P16], further comprising the step of contacting the cells with the fragmented extracellular matrix components in an aqueous medium before incubation. [P18] A method for promoting the differentiation of adipose-derived stem cells, comprising incubating cells containing at least adipose-derived stem cells in the presence of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. [P19] The method described in [P18], wherein the adipose-derived stem cells are derived from bovine tissue. [P20] The method described in [P18] or [P19], wherein incubation is performed for 96 hours or more and 384 hours or less. [P21] The method according to any one of [P18] to [P20], comprising incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. [P22] The method according to [P21], wherein the above-mentioned fragmented extracellular matrix component is a fragmented collagen component. [P23] The method according to [P21] or [P22], further comprising the step of contacting the cells with the fragmented extracellular matrix components in an aqueous medium before incubation. [Effects of the Invention]

[0008] According to the present invention, the differentiation of adipose-derived stem cells into mature adipocytes is promoted. By promoting differentiation into mature adipocytes, it becomes possible to produce a three-dimensional tissue containing mature adipocytes in a short time. [Brief explanation of the drawing]

[0009] [Figure 1]Figure 1 is a graph showing the results of comparing the fluorescence intensity of lipid staining with Nile Red on three-dimensional tissues prepared in Production Example 1 (*p<0.05, **p<0.01, ***p<0.001). [Figure 2] Figure 2 is a graph showing the results of comparing the fluorescence intensity of lipid staining with Nile Red on three-dimensional tissues prepared in Production Example 2 (*p<0.05, **p<0.01). [Figure 3] Figure 3 shows the results of lipid staining with Nile Red on the three-dimensional tissue prepared in Production Example 3. White dots indicate nuclei (Hoechst staining). [Figure 4] Figure 4 is a graph showing the results of comparing the rate of increase in fluorescence intensity of lipid staining with Nile Red on three-dimensional tissues prepared in Production Example 3. [Figure 5] Figure 5 shows the results of lipid staining with Nile Red on the three-dimensional tissue prepared in Production Example 4. The white dots indicate nuclei (Hoechst staining). [Figure 6] Figure 6 is a graph showing the results of comparing the rate of increase in fluorescence intensity of lipid staining with Nile Red on three-dimensional tissues prepared in Production Example 4. [Figure 7] Figure 7 is a graph showing a comparison of the fluorescence intensity of lipid staining with Nile Red on three-dimensional tissues at 3, 7, and 14 days of differentiation in Production Example 5. [Figure 8] Figure 8 shows the results of lipid staining with Nile Red on a three-dimensional tissue sample from production example 5, on day 3 of differentiation. White dots indicate nuclei (Hoechst staining). [Figure 9] Figure 9 shows the results of lipid staining with Nile Red on a three-dimensional tissue sample from production example 5 at day 7 of differentiation. White dots indicate nuclei (Hoechst staining). [Figure 10] Figure 10 shows the results of lipid staining with Nile Red on a three-dimensional tissue at 14 days of differentiation in production example 5. White dots indicate nuclei (Hoechst staining). [Figure 11] Figure 11 is a graph showing the results of comparing the fluorescence intensity of lipid staining with Nile Red on three-dimensional tissue samples at 7 days of differentiation in production example 6. [Modes for carrying out the invention]

[0010] The embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0011] In one embodiment, the present invention provides a method for producing a three-dimensional tissue containing mature adipocytes, comprising incubating cells containing at least adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor.

[0012] In this specification, "three-dimensional tissue" refers to a collection of cells (a cluster of cells) artificially created by cell culture in which the cells are arranged three-dimensionally. If the three-dimensional tissue contains extracellular matrix components as described later, the cells are arranged three-dimensionally via the extracellular matrix components. There are no particular restrictions on the shape of the three-dimensional tissue; for example, it can be sheet-like, spherical, nearly spherical, ellipsoidal, nearly ellipsoidal, hemispherical, nearly hemispherical, semicircular, nearly semicircular, rectangular prism-like, nearly rectangular prism-like, etc. Here, living tissue includes sweat glands, lymphatic vessels, sebaceous glands, etc., and its structure is more complex than that of the three-dimensional tissue. Therefore, three-dimensional tissue and living tissue can be easily distinguished.

[0013] In this specification, "cells" are not particularly limited, but may be cells derived from mammals such as monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats. The site of origin of the cells is also not particularly limited, and may be somatic cells derived from bone, muscle, internal organs, nerves, brain, skin, blood, etc., or germ cells. Furthermore, cells may be stem cells, or cultured cells such as primary cultured cells, subcultured cells, and cell line cells.

[0014] In this specification, "stem cells" means cells that have the ability to self-renew and multipotency. Stem cells include pluripotent stem cells, which have the ability to differentiate into any cell tumor, and tissue stem cells (also called somatic stem cells), which have the ability to differentiate into specific cell tumors. Examples of pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells). Examples of tissue stem cells include mesenchymal stem cells (e.g., adipose-derived stem cells, bone marrow-derived stem cells), hematopoietic stem cells, and neural stem cells.

[0015] The cells include at least adipose-derived stem cells. The origin of the adipose-derived stem cells is not particularly limited, and for example, stem cells collected from subcutaneous adipose tissue and epicardial adipose tissue may be used. When the three-dimensional tissue body containing mature adipocytes produced by the method of this embodiment is ultimately used to represent tissue in a specific part of a living organism, it is preferable to use stem cells derived from tissue corresponding to that part. Furthermore, the adipose-derived stem cells may be derived from, for example, cattle, horses, mice, rats, and pigs. According to the method of this embodiment, even when using cattle-derived adipose-derived stem cells, which are known to be particularly difficult to differentiate into mature adipocytes, differentiation into mature adipocytes can be promoted. Therefore, it is preferable to use cattle-derived adipose-derived stem cells because the effects of the present invention become more pronounced.

[0016] The cells may further contain cells other than adipose-derived stem cells. Examples of cells other than adipose-derived stem cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts; cancer cells such as colorectal cancer cells (e.g., human colorectal cancer cells (HT29)) and liver cancer cells; cardiomyocytes, epithelial cells (e.g., human gingival epithelial cells); lymphatic endothelial cells; nerve cells; dendritic cells; hepatocytes; adherent cells (e.g., immune cells); smooth muscle cells (e.g., aortic smooth muscle cells (Aorta-SMC)); pancreatic islet cells; and keratinocytes (e.g., human epidermal keratinocytes). However, since the present invention promotes the differentiation of adipose-derived stem cells into mature adipocytes, it is preferable that the cells containing at least adipose-derived stem cells, i.e., the cells before incubation, do not contain mature adipocytes, as this makes the effects of the present invention more pronounced.

[0017] The three-dimensional tissue in this embodiment includes mature adipocytes. In the three-dimensional tissue including mature adipocytes, it is preferable that 90% or more of the total number of adipocytes are mature adipocytes, and more preferably that all of them are mature adipocytes. In this specification, "adipocytes" means all adipocytes except adipose-derived stem cells, and includes mature adipocytes and adipocytes not included in adipose-derived stem cells.

[0018] The size of lipid droplets can be used as an indicator of the maturity of adipocytes. Lipid droplets are intracellular organelles that store lipids such as triglycerides and cholesterol, and have a droplet-like shape because these lipids are covered by a single membrane of phospholipids. Furthermore, the surface of these phospholipids shows expression of proteins specific to adipose tissue (such as perilipin). Although there is variability in the size of lipid droplets in mature adipocytes, for example, if the average size of lipid droplets is 20 μm or more, the adipocytes can be considered to be mature to a certain extent, i.e., mature adipocytes.

[0019] The adipocyte content may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more relative to the total number of cells in the three-dimensional tissue, and may be 95% or less, 90% or less, 80% or less, or 75% or less. Furthermore, the mature adipocyte content may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more relative to the total number of cells in the three-dimensional tissue, and may be 95% or less, 90% or less, 80% or less, or 75% or less.

[0020] The three-dimensional tissue may further contain cells other than mature adipocytes. Examples of cells other than mature adipocytes include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts; cancer cells such as colorectal cancer cells (e.g., human colorectal cancer cells (HT29)) and liver cancer cells; cardiomyocytes, epithelial cells (e.g., human gingival epithelial cells); lymphatic endothelial cells; nerve cells; dendritic cells; hepatocytes; adherent cells (e.g., immune cells); smooth muscle cells (e.g., aortic smooth muscle cells (Aorta-SMC)); pancreatic islet cells; and keratinocytes (e.g., human epidermal keratinocytes). However, since the present invention promotes the differentiation of adipose-derived stem cells into mature adipocytes, the effects of the present invention become more pronounced. Therefore, in the three-dimensional tissue after incubation, it is preferable that adipose-derived stem cells constitute 10% or less of the total number of cells in the three-dimensional tissue, more preferably 5% or less, and even more preferably that the tissue does not contain adipose-derived stem cells.

[0021] The three-dimensional tissue may have a vascular network between cells. The presence of a vascular network between cells is expected to allow for long-term maintenance of the three-dimensional tissue and to facilitate engraftment when transplanted into mammals or other organisms.

[0022] "Having a vascular network between cells" means that, similar to living tissue, the tissue has a structure in which branched blood vessels extend between cells, surrounding them. Whether or not a vascular network similar to that of living tissue is formed can be determined, for example, based on the diversity of the number of blood vessel branches and / or the length between branches and / or the diameter of blood vessels in living tissue. For example, if the average number of blood vessel branches in a three-dimensional tissue is between 80% and 150%, between 85% and 130%, or between 90% and 120% of the average number of blood vessel branches in living tissue, it may be judged to be similar to the number of blood vessel branches in living tissue. Alternatively, for example, if the average number of blood vessel branches in a three-dimensional tissue is between 2.5 and 4.5, or between 3.0 and 4.2, it may be judged to be similar to the number of blood vessel branches in living tissue. For example, if the average length between branching vessels in a three-dimensional tissue is 80% to 150%, 85% to 130%, and 90% to 120% of the average length between branching vessels in living tissue, it may be judged to be similar to the length between branching vessels in living tissue. In living tissue, both large and small blood vessels are observed. Therefore, for example, if both large vessels (e.g., 10 μm to less than 25 μm) and small vessels (e.g., greater than 0 μm and less than 10 μm) are observed, similar to living tissue, it may be judged to have a diversity similar to the diameter of blood vessels in living tissue. Also, for example, if 60% or more, 70% or more, or 80% or more of the total blood vessel diameter is distributed in the range greater than 0 μm and less than 25 μm, it may be judged to have a diversity similar to the diameter of blood vessels in living tissue. It is preferable that a three-dimensional tissue containing mature adipocytes has a vascular network between adipocytes. In that case, it is preferable that not only does it have a vascular network, but the adipocytes surrounded by the blood vessels are also similar to those in living tissue. For example, if the average size of lipid droplets in the adipocytes in the three-dimensional tissue according to this embodiment is 20 μm to 180 μm, or 100 μm to 180 μm, it may be determined that the three-dimensional tissue has adipocytes similar to those in living tissue.When comparing the above-mentioned biological tissues and three-dimensional tissues, the comparison should be made under the same conditions (for example, per unit volume, per unit area in the case of image analysis, per unit sample, etc.).

[0023] The thickness of the three-dimensional tissue is preferably 10 μm or more, more preferably 100 μm or more, and even more preferably 1000 μm or more. Such a three-dimensional tissue has a structure closer to living tissue and is suitable as a substitute for experimental animals and as a transplant material. There is no particular upper limit to the thickness of the three-dimensional tissue, but for example it may be 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0024] Here, "thickness of the three-dimensional structure" refers to the distance between the two ends in a direction perpendicular to the main surface, if the three-dimensional structure is in the form of a sheet or a rectangular parallelepiped. If the main surface has irregularities, the thickness refers to the distance at the thinnest part of the main surface.

[0025] Furthermore, if the three-dimensional structure is spherical or nearly spherical, it refers to its diameter. Moreover, if the three-dimensional structure is ellipsoidal or nearly ellipsoidal, it refers to its minor axis. If the three-dimensional structure is nearly spherical or nearly ellipsoidal and has irregularities on its surface, the thickness refers to the shortest distance between two points where a line passing through the centroid of the three-dimensional structure intersects the surface.

[0026] The method of this embodiment includes incubating cells containing at least adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor. Incubation in the presence of a TGFβ type I receptor inhibitor promotes the differentiation of adipose-derived stem cells into mature adipocytes. In the method of this embodiment, by promoting differentiation while the adipose-derived stem cells are contained within a three-dimensional tissue, or by promoting differentiation in a situation where a three-dimensional tissue is formed so that the adipose-derived stem cells are contained within the tissue, a three-dimensional tissue containing vascular cells can ultimately be produced. Therefore, incubating cells containing at least adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor may involve incubating only the cells in the presence of the TGFβ type I receptor inhibitor, incubating a mixture of cells and extracellular matrix components in the presence of the TGFβ type I receptor inhibitor, or incubating a three-dimensional tissue containing cells and extracellular matrix components in the presence of the TGFβ type I receptor inhibitor after it has been formed.

[0027] Examples of TGFβ type I receptor inhibitors include inhibitors of TGFβR1 kinases, namely ALK5, ALK2, ALK4, and ALK7. The TGFβ type I receptor inhibitor is not particularly limited as long as it has an inhibitory effect on the TGFβ type I receptor. Examples of ALK5, ALK4, and ALK7 inhibitors include 4-[4-(1,3-benzodioxyol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide, and examples of ALK5 inhibitors include 2-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]-1,5-naphthyridine, 2-(3-(6-methylpyridinyl-2-yl)-1H-pyrazole-4-yl)-1,5-naphthyridine hydrochloride, and N-(2-fluorophenyl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methyl-2-pyridyl)-1H-imidazole-2-methanamine.

[0028] Incubating in the presence of a TGFβ type I receptor inhibitor may, for example, mean incubating (cultivating) cells containing at least adipose-derived stem cells in a culture medium containing a TGFβ type I receptor inhibitor.

[0029] The method of this embodiment may further include incubating cells containing at least adipose-derived stem cells in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. Alternatively, the cells may be incubated in the presence of two or more, three or more, four or more, five or more, or six or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid, or in the presence of elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. Incubating in the presence of these fatty acids further promotes the differentiation of adipose-derived stem cells into mature adipocytes. Incubation in the presence of the above fatty acids may or may not be performed simultaneously with incubation in the presence of a TGFβ type I receptor inhibitor. If performed simultaneously, for example, the above fatty acids may be included in the culture medium together with the TGFβ type I receptor inhibitor. In this case, for example, a culture medium containing the above fatty acids and the TGFβ type I receptor inhibitor may be used beforehand, or the TGFβ type I receptor inhibitor may be added to the culture medium containing the above fatty acids, or the above fatty acids may be added to the culture medium containing the TGFβ type I receptor inhibitor. If performed not simultaneously, for example, cells may be incubated in a culture medium containing the above fatty acids and then incubated in a culture medium containing the TGFβ type I receptor inhibitor, or cells may be incubated in a culture medium containing the TGFβ type I receptor inhibitor and then incubated in a culture medium containing the above fatty acids.

[0030] The medium is not particularly limited, and a suitable medium can be selected according to the type of cells to be cultured. The medium may be a solid medium or a liquid medium, but a liquid medium is preferred. Examples of the medium include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. Further, the liquid medium may be a gel-like one such as fibrin gel, hydrogel, Matrigel, collagen gel, and gelatin gel. Cells may be added to the gel-like liquid medium, or the liquid medium containing cells may be gelled and used. The medium may be a medium supplemented with serum or a serum-free medium. The medium may be a mixed medium in which two types of media are mixed.

[0031] The amount of the TGFβ type I receptor inhibitor may be an amount sufficient for each adipose-derived stem cell to contact the inhibitor. For example, for 1×10 6 cells of adipose-derived stem cells, it may be 2.0×10 -11 mol to 2.0×10 -8 mol, or 1.0×10 -9 mol to 2.0×10 -8 mol. For example, for 5×10 6 cells of adipose-derived stem cells, it may be 1.0×10 -10 mol to 1.0×10 -7 mol, or 5.0×10 -9 mol to 1.0×10 -7 mol. Further, the TGFβ type I receptor inhibitor may be, for example, 6.0 ng to 6.0 μg, or 300 ng to 6.0 μg for 1×10 6 cells of adipose-derived stem cells. For example, for 5×10 6 cells of adipose-derived stem cells, it may be 30 ng to 30 μg, or 1500 ng to 30 μg.

[0032] When cells containing at least adipose-derived stem cells are incubated in a medium containing a TGFβ type I receptor inhibitor, the content of the TGFβ type I receptor inhibitor in the medium may be, for example, 0.1 μM to 100 μM, 0.5 μM to 50 μM, 1 μM to 10 μM, or 2 μM to 8 μM, or 0.5 μM or more, 0.7 μM or more, 1 μM or more, 2 μM or more, 3 μM or more, 4 μM or more, 5 μM or more, or 20 μM or less, 15 μM or less, 12 μM or less, 10 μM or less, 8 μM or less, or 7 μM or less.

[0033] When cells containing at least adipose-derived stem cells are incubated in a culture medium containing the above fatty acids, the content of each fatty acid in the culture medium may be, for example, 0.1 μM to 200 μM, 2 μM to 100 μM, 10 μM to 60 μM, 30 μM to 50 μM, 30 μM to 150 μM, or 80 μM to 120 μM, or 1 μM or more, 5 or more, 10 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, 50 μM or more, 60 μM or more, 70 μM or more, 80 μM or more, 90 μM or more, or 200 μM or less, 150 μM or less, 120 μM or less, 100 μM or less, 80 μM or less, or 70 μM or less.

[0034] Each fatty acid, for example, in adipose-derived stem cells 1 × 10 6 For cells, 2.0 × 10 -11 mol ~ 4.0 × 10 -8 mol, or 1.0 × 10⁻⁶ -9 mol ~ 2.0 × 10 -8 The number of moles may be used. The weight of each fatty acid is, for example, 1 × 10⁻⁶ of adipose-derived stem cells. 6 The dosage may be 6 ng to 15 μg or 250 ng to 10 μg per cell.

[0035] The cell density in the culture medium before incubation can be appropriately determined according to the shape, thickness, and size of the culture vessel of the target three-dimensional tissue. For example, the cell density in the culture medium can be 1 to 10 8 It may be cells / mL, 10 3 ~10 7It may be cells / mL.

[0036] Incubation is not particularly restricted and can be carried out under conditions suitable for the type of cells being cultured. For example, the incubation temperature may be 20°C to 40°C or 30°C to 37°C. The pH of the culture medium may be 6 to 8 or 7.2 to 7.4. The incubation time may be 24 hours to 336 hours, 72 hours to 336 hours, 96 hours to 384 hours, or 96 hours to 288 hours. The incubation time after adding the TGFβ type I receptor inhibitor and / or the above fatty acids may be 24 hours to 336 hours, 72 hours to 336 hours, 96 hours to 384 hours, or 96 hours to 288 hours.

[0037] The culture vessel (support) used for culturing cells is not particularly limited and may be, for example, a well insert, a low-adhesion plate, or a plate with a bottom shape such as U-shaped or V-shaped. The cells may be cultured while attached to the support, or without being attached to the support, or they may be separated from the support during culture. When culturing cells without being attached to the support, or when separating them from the support during culture, it is preferable to use a plate with a bottom shape such as U-shaped or V-shaped that inhibits cell adhesion to the support, or a low-adhesion plate.

[0038] The method of this embodiment may include incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of a TGFβ type I receptor inhibitor. By incubating with fragmented extracellular matrix components, a three-dimensional tissue can be obtained in which cells are arranged three-dimensionally via the fragmented extracellular matrix components. It is preferable that the three-dimensional tissue is in which the fragmented extracellular matrix components are arranged in the gaps between the cells. The cells between cells may be homogeneous or heterogeneous. When using a culture medium containing a TGFβ type I receptor inhibitor, the fragmented extracellular matrix components may be added to the medium at the same time as the TGFβ type I receptor inhibitor, before the TGFβ type I receptor inhibitor, or after the TGFβ type I receptor inhibitor.

[0039] Fragmented extracellular matrix components can be obtained by fragmenting extracellular matrix components. In this specification, "extracellular matrix component" refers to an aggregate of extracellular matrix molecules formed by multiple extracellular matrix molecules. The extracellular matrix refers to substances that exist outside the cell in an organism. Any substance can be used as the extracellular matrix, as long as it does not adversely affect cell growth and the formation of cell aggregates. Specific examples include, but are not limited to, collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, tenascin, entactin, and fibrillin. Extracellular matrix components may be used individually or in combination. Extracellular matrix components may, for example, contain collagen components, or may be collagen components. In this embodiment, it is preferable that the extracellular matrix component is a substance that exists outside animal cells, i.e., an animal extracellular matrix component.

[0040] The extracellular matrix molecule may be a modified or variant of the extracellular matrix molecule described above, or a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect cell growth and cell aggregate formation. The extracellular matrix molecule may have a repeating sequence represented by Gly-XY, which is characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. Multiple Gly-XY sequences may be identical or different. Having a repeating sequence represented by Gly-XY reduces constraints on the arrangement of the molecular chain, resulting in improved functionality as a scaffold material during cell culture, for example. In an extracellular matrix molecule having a repeating sequence represented by Gly-XY, the proportion of the sequence represented by Gly-XY may be 80% or more of the total amino acid sequence, preferably 95% or more. The extracellular matrix molecule may also be a polypeptide having an RGD sequence. An RGD sequence refers to a sequence represented as Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). The presence of an RGD sequence further promotes cell adhesion, making it more suitable, for example, as a scaffold material during cell culture. Examples of extracellular matrix molecules containing both a Gly-XY sequence and an RGD sequence include collagen, fibronectin, vitronectin, laminin, and cadherin.

[0041] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. An example of non-fibrous collagen is type IV collagen.

[0042] Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0043] The extracellular matrix components may include at least one selected from the group consisting of collagen, laminin, and fibronectin, as this enhances the effects of the present invention, and it is preferable that they include collagen. The collagen is preferably fibrous collagen, and more preferably type I collagen. Commercially available collagen may be used as the fibrous collagen, and a specific example of this is porcine skin-derived type I collagen manufactured by Nippon Ham Co., Ltd.

[0044] The extracellular matrix components may be extracellular matrix components derived from animals. Examples of animal species from which the extracellular matrix components may be derived include, but are not limited to, humans, pigs, and cattle. The extracellular matrix components may be components derived from one type of animal, or components derived from multiple types of animals may be used in combination.

[0045] "Fragmentation" refers to reducing the size of aggregates of extracellular matrix molecules. Fragmentation may be carried out under conditions that cleave bonds within extracellular matrix molecules, or under conditions that do not cleave bonds within extracellular matrix molecules. Unlike enzymatic treatment, the molecular structure of extracellular matrix fragmented by the application of physical force usually does not change from before fragmentation (the molecular structure is maintained). Fragmented extracellular matrix components may include defibrated extracellular matrix components (defibrated extracellular matrix components), which are components obtained by defibrating the above-mentioned extracellular matrix components by the application of physical force. Defibration is a form of fragmentation, for example, carried out under conditions that do not cleave bonds within extracellular matrix molecules.

[0046] There are no particular limitations on the method for fragmenting extracellular matrix components. For example, extracellular matrix components may be defibrated by applying physical force, such as using an ultrasonic homogenizer, agitator homogenizer, or high-pressure homogenizer. When using an agitator homogenizer, the extracellular matrix components may be homogenized directly, or they may be homogenized with an aqueous medium such as physiological saline or an organic solvent such as ethanol. Furthermore, by adjusting the homogenization time and number of repetitions, it is possible to obtain defibrated extracellular matrix components of millimeter or nanometer size. Defibrated extracellular matrix components can also be obtained by repeated freeze-thaw cycles.

[0047] The fragmented extracellular matrix component may contain at least a portion of the defibrated extracellular matrix component. Alternatively, the fragmented extracellular matrix component may consist solely of the defibrated extracellular matrix component. In other words, the fragmented extracellular matrix component may be the defibrated extracellular matrix component. The defibrated extracellular matrix component preferably contains the defibrated collagen component (defibrated collagen component). The defibrated collagen component preferably maintains the triple helix structure derived from collagen. The defibrated collagen component may be a component that partially maintains the triple helix structure derived from collagen.

[0048] Examples of the shape of fragmented extracellular matrix components include fibrous structures. Fibrous refers to a shape composed of thread-like collagen components, or a shape composed of thread-like extracellular matrix components cross-linked between molecules. At least a portion of the fragmented extracellular matrix components may be fibrous. Fibrous extracellular matrix components include thin threads (fibrillaries) formed by the aggregation of multiple thread-like extracellular matrix molecules, threads formed by further aggregation of fibrillaries, and defibrillated versions of these threads. In fibrous extracellular matrix components, the RGD sequence is preserved without disruption.

[0049] The average length of the fragmented extracellular matrix components may be between 100 nm and 400 μm, or between 100 nm and 200 μm. In one embodiment, the average length of the fragmented extracellular matrix components may be between 5 μm and 400 μm, between 10 μm and 400 μm, between 22 μm and 400 μm, or between 100 μm and 400 μm. In another embodiment, from the viewpoint of 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 most of the fragmented extracellular matrix components is within the above numerical range. Specifically, it is preferable that the average length of 95% of the fragmented extracellular matrix components is within the above numerical range. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average length within the above range, and more preferably a defibrated collagen component having an average length within the above range.

[0050] The average diameter of the fragmented extracellular matrix components may be 10 nm to 30 μm, 30 nm to 30 μm, 50 nm to 30 μm, 100 nm to 30 μm, 1 μm to 30 μm, 2 μm to 30 μm, 3 μm to 30 μm, 4 μm to 30 μm, or 5 μm to 30 μm. The fragmented extracellular matrix components are preferably fragmented collagen components with an average diameter within the above range, and more preferably defibrated collagen components with an average diameter within the above range.

[0051] 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 performing image analysis. In this specification, "average length" refers to the average value of the length in the longitudinal direction of the measured sample, and "average diameter" refers to the average value of the length in the direction perpendicular to the longitudinal direction of the measured sample.

[0052] The fragmented extracellular matrix component may include, for example, a fragmented collagen component, or may consist of a fragmented collagen component. "Fragmented collagen component" means a collagen component, such as a fibrous collagen component, that has been fragmented and maintains a triple helix structure. The average length of the fragmented collagen component is preferably 100 nm to 200 μm, more preferably 22 μm to 200 μm, and even more preferably 100 μm to 200 μm. The average diameter of the fragmented collagen component is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0053] At least a portion of the fragmented extracellular matrix components may be cross-linked intermolecularly or intramolecularly. The extracellular matrix components may be cross-linked intramolecularly or between the extracellular matrix molecules that constitute the extracellular matrix components.

[0054] Methods for crosslinking include, for example, physical crosslinking by applying heat, ultraviolet light, or radiation, and chemical crosslinking by using crosslinking agents or enzymatic reactions, but the method is not particularly limited. From the viewpoint of not hindering cell growth, physical crosslinking is preferred. Crosslinking (physical crosslinking and chemical crosslinking) may be crosslinking via covalent bonds.

[0055] When the extracellular matrix components include collagen components, crosslinking may occur between collagen molecules (triple helix structure) or between collagen fibrils formed by collagen molecules. Crosslinking may be thermal crosslinking. Thermal crosslinking can be performed, for example, by heat treatment under reduced pressure using a vacuum pump. When thermal crosslinking of collagen components is performed, the extracellular matrix components may be crosslinked by the amino groups of collagen molecules forming peptide bonds (-NH-CO-) with the carboxyl groups of the same or other collagen molecules.

[0056] Extracellular matrix components can also be crosslinked using a crosslinking agent. The crosslinking agent may be, for example, one that can crosslink carboxyl groups with amino groups, or one that can crosslink amino groups with each other. From the viewpoint of economy, safety, and ease of handling, aldehyde-based, carbodiimide-based, epoxide-based, and imidazole-based crosslinking agents are preferred, for example. Specifically, water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide sulfonate can be mentioned.

[0057] The degree of crosslinking can be appropriately selected depending on the type of extracellular matrix component, the crosslinking method, etc. The degree of crosslinking may be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, and may also be 30% or less, 20% or less, or 15% or less. By having the degree of crosslinking within the above range, the extracellular matrix molecules can be appropriately dispersed, and the redispersibility after dry storage is good.

[0058] When amino groups in extracellular matrix components are used for crosslinking, the degree of crosslinking can be quantified using the TNBS (trinitrobenzenesulfonic acid) method. The degree of crosslinking obtained by the TNBS method may be within the range described above. The degree of crosslinking obtained by the TNBS method is the proportion of amino groups used for crosslinking out of the amino groups present in the extracellular matrix. When the extracellular matrix components include collagen components, it is preferable that the degree of crosslinking measured by the TNBS method is within the range described above.

[0059] The degree of crosslinking may be calculated by quantifying the carboxyl groups. For example, in the case of water-insoluble extracellular matrix components, it may be quantified by the TBO (toluidine blue O) method. The degree of crosslinking obtained by the TBO method may be within the range described above.

[0060] The extracellular matrix component content in the three-dimensional tissue may be 0.01 to 90% by mass, preferably 10 to 90% by mass, preferably 10 to 80% by mass, preferably 10 to 70% by mass, preferably 10 to 60% by mass, preferably 1 to 50% by mass, preferably 10 to 50% by mass, more preferably 10 to 30% by mass, and even more preferably 20 to 30% by mass.

[0061] Here, "extracellular matrix components in a three-dimensional tissue" refers to the extracellular matrix components that constitute the three-dimensional tissue, and may originate from endogenous extracellular matrix components or from exogenous extracellular matrix components.

[0062] "Endogenous extracellular matrix components" refers to extracellular matrix components produced by extracellular matrix-producing cells. Examples of extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, as mentioned above. Endogenous extracellular matrix components may be fibrous or non-fibrous.

[0063] "Exogenous extracellular matrix components" refer to extracellular matrix components supplied from an external source. The animal species from which these exogenous extracellular matrix components originate may be the same as or different from that of endogenous extracellular matrix components. Examples of originating animal species include humans, pigs, and cattle. Furthermore, exogenous extracellular matrix components may be artificial extracellular matrix components.

[0064] When the extracellular matrix component is collagen, the exogenous extracellular matrix component is also called "exogenous collagen component." "Exogenous collagen component," which refers to collagen component supplied from outside, is an aggregate of collagen molecules formed by multiple collagen molecules, and specifically includes fibrous collagen and non-fibrous collagen. The exogenous collagen component is preferably fibrous collagen. The above-mentioned fibrous collagen refers to the collagen component that is the main component of collagen fibers, and examples include type I collagen, type II collagen, and type III collagen. The above-mentioned fibrous collagen may be commercially available collagen, and a specific example is type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. An example of exogenous non-fibrous collagen is type IV collagen.

[0065] In the case of exogenous extracellular matrix components, the animal species from which they originate may differ from that of the cells. Furthermore, if the cells include extracellular matrix-producing cells, the animal species from which the exogenous extracellular matrix components originate may differ from those of the extracellular matrix-producing cells. In other words, exogenous extracellular matrix components may be heterogeneous extracellular matrix components.

[0066] In other words, when a three-dimensional tissue contains endogenous extracellular matrix components and fragmented extracellular matrix components, the extracellular matrix component content of the three-dimensional tissue refers to the total amount of endogenous extracellular matrix components and fragmented extracellular matrix components. The extracellular matrix content can be calculated from the volume of the obtained three-dimensional tissue and the mass of the decellularized three-dimensional tissue.

[0067] For example, if the extracellular matrix component contained in a three-dimensional tissue is collagen, a method for quantifying the amount of collagen in the three-dimensional tissue may be the following method for quantifying hydroxyproline. A sample is prepared by mixing hydrochloric acid (HCl) with a dissolution containing the three-dimensional tissue, incubating at a high temperature for a predetermined time, returning to room temperature, and diluting the supernatant obtained by centrifugation to a predetermined concentration. A hydroxyproline standard solution is prepared by processing it in the same way as the sample, and then diluting it stepwise. The sample and standard are each subjected to the predetermined treatment with hydroxyproline assay buffer and detection reagent, and the absorbance at 570 nm is measured. The amount of collagen is calculated by comparing the absorbance of the sample with that of the standard. Alternatively, the three-dimensional tissue may be directly suspended and dissolved in high-concentration hydrochloric acid, the dissolution is centrifuged, and the supernatant is collected and used for collagen component quantification. Furthermore, the three-dimensional tissue to be dissolved may be in the state as recovered from the culture medium, or it may be dried after recovery to remove liquid components before dissolution. However, when quantifying collagen components by dissolving a three-dimensional tissue in its as-recovered state from the culture medium, the measured weight of the three-dimensional tissue is expected to vary due to the influence of culture medium components absorbed by the three-dimensional tissue and residual culture medium due to problems with the experimental procedure. Therefore, from the viewpoint of stably measuring the weight of the structure and the amount of collagen component per unit weight, it is preferable to use the weight after drying as the basis.

[0068] More specifically, the following methods can be used to quantify the amount of collagen.

[0069] (Sample preparation) The entire volume of the freeze-dried three-dimensional tissue is mixed with 6 mol / L HCl and incubated in a heat block at 95°C for at least 20 hours, then allowed to return to room temperature. After centrifugation at 13000 g for 10 minutes, the supernatant of the sample solution is collected. After diluting with 6 mol / L HCl as appropriate so that the results fall within the calibration curve in the measurement described later, 200 μL is diluted with 100 μL of ultrapure water to prepare the sample. 35 μL of the sample is used.

[0070] (Standard preparation) Add 125 μL of standard solution (1200 μg / mL in acetic acid) and 125 μL of 12 mol / L HCl to a screw-cap tube and mix. Incubate at 95°C for 20 hours on a heat block, then return to room temperature. Centrifuge at 13000 g for 10 minutes, then dilute the supernatant with ultrapure water to prepare S1 (300 μg / mL). Dilute S1 stepwise to prepare S2 (200 μg / mL), S3 (100 μg / mL), S4 (50 μg / mL), S5 (25 μg / mL), S6 (12.5 μg / mL), and S7 (6.25 μg / mL). Prepare S8 (0 μg / mL) using only 90 μL of 4 mol / L HCl.

[0071] (assay) Add 35 μL each of the standard and sample to a plate (included in the QuickZyme Total Collagen Assay Kit, QuickZyme Biosciences). Add 75 μL of assay buffer (included in the kit) to each well. Seal the plate and incubate at room temperature for 20 minutes while shaking. Remove the seal and add 75 μL of detection reagent (reagent A:B = 30 μL:45 μL, included in the kit) to each well. Seal the plate, mix the solutions by shaking, and incubate at 60°C for 60 minutes. Cool thoroughly on ice, remove the seal, and measure the absorbance at 570 nm. Calculate the amount of collagen component by comparing the absorbance of the sample with that of the standard.

[0072] The collagen component in a three-dimensional tissue may be defined by its area ratio or volume ratio. "Defining by area ratio or volume ratio" means, for example, making the collagen component in the three-dimensional tissue distinguishable from other tissue components using known staining methods (e.g., immunohistochemical staining using anti-collagen antibodies, or Masson's trichrome staining), and then calculating the ratio of the collagen component's area to the entire three-dimensional tissue using macroscopic observation, various microscopes, and image analysis software. When defining by area ratio, the method of defining the area ratio is not limited to any specific cross-section or surface within the three-dimensional tissue; however, if the three-dimensional tissue is spherical, for example, it may be defined by a cross-sectional view passing through its approximate center.

[0073] For example, when defining the collagen component in a three-dimensional tissue by area ratio, the area ratio is preferably 0.01 to 99%, 1 to 99%, 5 to 90%, 7 to 90%, 20 to 90%, and more preferably 50 to 90%, based on the total area of ​​the three-dimensional tissue. The "collagen component in a three-dimensional tissue" is as described above. The area ratio of the collagen component constituting the three-dimensional tissue refers to the area ratio of the endogenous collagen component and the exogenous collagen component combined. The area ratio of the collagen component can be calculated, for example, by staining the obtained three-dimensional tissue with Masson's trichrome and taking the ratio of the area of ​​the blue-stained collagen component to the total area of ​​the cross-section passing through approximately the center of the three-dimensional tissue.

[0074] The three-dimensional tissue is preferably retained at a concentration of 0.25% trypsin, at a temperature of 37°C, pH 7.4, and reaction time of 15 minutes, with a retention rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more. Such a three-dimensional tissue is stable and less susceptible to enzymatic degradation during or after culture. The above retention rate can be calculated, for example, from the mass of the three-dimensional tissue before and after trypsin treatment.

[0075] The above three-dimensional tissue may have a residual rate of 70% or more after collagenase treatment at a collagenase concentration of 0.25%, a temperature of 37°C, a pH of 7.4, and a reaction time of 15 minutes, more preferably 80% or more, and even more preferably 90% or more. Such a three-dimensional tissue is stable and less susceptible to enzymatic degradation during or after culture.

[0076] Incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of a TGFβ type I receptor inhibitor means, for example, incubating (cultivating) the cells in a medium containing a TGFβ type I receptor inhibitor and fragmented extracellular matrix components. In this case, for example, a medium containing fragmented extracellular matrix components and a TGFβ type I receptor inhibitor beforehand may be used, or a TGFβ type I receptor inhibitor may be added to a medium containing fragmented extracellular matrix components, or a fragmented extracellular matrix component may be added to a medium containing a TGFβ type I receptor inhibitor.

[0077] The process may further include a step of contacting the cells with the fragmented extracellular matrix components in an aqueous medium before incubation. In this case, the TGFβ type I receptor inhibitor and / or the fatty acid described above may be present in the aqueous medium, added before incubation, or added during incubation. The aqueous medium may be the same as or different from the one used during incubation. By incubating the cells with the fragmented extracellular matrix components in an aqueous medium, the fragmented extracellular matrix components are positioned in the gaps between the cells, and a three-dimensional tissue can be produced in which the cells and fragmented extracellular matrix components are uniformly distributed three-dimensionally throughout the entire three-dimensional tissue. For example, after incubating the cells with the fragmented extracellular matrix components in an aqueous medium, further incubation may be performed in the presence of the TGFβ type I receptor inhibitor and / or the fatty acid. The incubation time for contacting the cells with the fragmented extracellular matrix components in an aqueous medium may be, for example, 12 to 72 hours.

[0078] An "aqueous medium" refers to a liquid in which water is an essential component. There are no particular restrictions on the aqueous medium as long as it can stably contain fragmented extracellular matrix components. For example, examples of aqueous mediums include, but are not limited to, salines such as phosphate-buffered saline (PBS), Dulbecco's Modified Eagle medium (DMEM), and vascular endothelial cell medium (EGM2).

[0079] The pH of the aqueous medium is preferably within a range that does not adversely affect cell growth and cell aggregate formation. From the viewpoint of reducing the burden on cells when introduced to cells, the pH of the aqueous medium may be, for example, 7.0 or higher and 8.0 or lower. Specifically, the pH of the aqueous medium 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 aqueous medium preferably has buffering capacity within the above pH range, and more preferably is a liquid medium. There are no particular restrictions on the liquid medium, and a suitable medium can be selected depending on the type of cells to be cultured. Examples of such media include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The medium may be a serum-added medium or a serum-free medium. Furthermore, the liquid medium may be a mixed medium obtained by mixing two or more types of media.

[0080] By dispersing fragmented extracellular matrix components in an aqueous medium, they become more readily accessible to cells in the aqueous medium, potentially promoting the formation of three-dimensional tissues. The contact process may involve, but is not limited to, methods such as: mixing an aqueous medium containing fragmented extracellular matrix components with an aqueous medium containing cells; adding cells to an aqueous medium containing fragmented extracellular matrix components; adding an aqueous medium containing extracellular matrix components to a culture medium containing cells; adding cells to an aqueous medium containing extracellular matrix components; or adding extracellular matrix components and cells to a pre-prepared aqueous medium, respectively.

[0081] The concentration of fragmented extracellular matrix components in the contact step can be appropriately determined according to the shape, thickness, and size of the culture vessel of the target three-dimensional tissue. For example, the concentration of fragmented extracellular matrix components in the aqueous medium during the contact step may be 0.1 to 90% by mass or 1 to 30% by mass.

[0082] The amount of fragmented extracellular matrix components in the contact process is, for example, 1.0 × 10⁻⁶. 6 The amount relative to the cells may be 0.1-100 mg, 0.5-50 mg, 0.8-25 mg, 1.0-10 mg, 1.0-5.0 mg, 1.0-2.0 mg, or 1.0-1.8 mg, and may be 0.7 mg or more, 1.1 mg or more, 1.2 mg or more, 1.3 mg or more, or 1.4 mg or more, and may be 7.0 mg or less, 3.0 mg or less, 2.3 mg or less, 1.8 mg or less, 1.7 mg or less, 1.6 mg or less, or 1.5 mg or less.

[0083] In the contact process, the mass ratio of fragmented extracellular matrix components to cells (fragmented extracellular matrix components / cells) is preferably 1 / 1 to 1000 / 1, more preferably 9 / 1 to 900 / 1, and even more preferably 10 / 1 to 500 / 1.

[0084] Furthermore, the amount of fragmented extracellular matrix components in the contact step may be 0.1% to 10% by weight, 0.5% to 8% by weight, or 1% to 5% by weight relative to the total weight of the culture medium.

[0085] One embodiment of a method for producing a three-dimensional tissue may include mixing fibrinogen and thrombin simultaneously or separately during a contact step, or after the contact step but before incubation. By mixing fibrinogen and thrombin, they react to form fibrin. The fibrinogen content may be, for example, 1 to 10 mg / mL, 2 to 8 mg / mL, or 5 to 6 mg / mL relative to the culture medium.

[0086] The process may further include a step of settling the fragmented extracellular matrix components and cells together in an aqueous medium after the contact step and before incubation. Performing such a step results in a more uniform distribution of the fragmented extracellular matrix components and cells in the three-dimensional tissue. There are no particular limitations on the specific method, but one example is to centrifuge the culture medium containing the fragmented extracellular matrix components and cells.

[0087] The cell density in the culture medium before incubation, as described above, may be the same as the cell density in the aqueous medium during the contact step.

[0088] As one embodiment, the present invention also provides a method for promoting the differentiation of adipose-derived stem cells, comprising incubating cells containing at least adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor. As described above, the differentiation of adipose-derived stem cells into mature adipocytes can be promoted by incubating adipose-derived stem cells in the presence of a TGFβ type I receptor inhibitor. The method of this embodiment can also be used in the production of tissues, which may be three-dimensional or two-dimensional cultures. The method of this embodiment includes promoting differentiation while adipose-derived stem cells are contained in a tissue, or promoting differentiation in a situation in which a tissue is formed so that adipose-derived stem cells are contained in the tissue, thereby ultimately producing a tissue containing mature adipocytes.

[0089] The method of this embodiment can be applied not only to three-dimensional culture in the production of three-dimensional tissues described above, but also to two-dimensional culture, since it is sufficient that differentiation is promoted by contact of at least some adipose-derived stem cells with a TGFβ type I receptor inhibitor.

[0090] Furthermore, the method of this embodiment may also be a method for promoting the differentiation of adipose-derived stem cells in the method for producing a three-dimensional tissue described above.

[0091] The specific aspects of the method of this embodiment, including the three-dimensional tissue, cells, incubation, fragmented extracellular matrix components, and each step, can be applied without limitation to the specific aspects described above. For example, the method of this embodiment may also include a step of bringing the cells and the fragmented extracellular matrix components into contact in an aqueous medium before incubation.

[0092] The present invention also provides, as one embodiment, a method for promoting the differentiation of adipose-derived stem cells, comprising incubating cells containing at least adipose-derived stem cells in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. Furthermore, as one embodiment, a method for promoting the differentiation of adipose-derived stem cells is also provided, comprising incubating cells containing at least adipose-derived stem cells in the presence of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. By incubating adipose-derived stem cells in the presence of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid, the differentiation of adipose-derived stem cells into mature adipocytes can be promoted.

[0093] The method of this embodiment may include incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid, or incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. By incubating with fragmented extracellular matrix components, a three-dimensional tissue can be obtained in which cells are arranged three-dimensionally via the fragmented extracellular matrix components. It is preferable that the three-dimensional tissue is such that the fragmented extracellular matrix components are arranged in the gaps between the cells.

[0094] The specific aspects of the method of this embodiment, including the three-dimensional tissue, cells, incubation, fragmented extracellular matrix components, and each step, can be applied without limitation to the specific aspects described above. For example, the method of this embodiment may also include a step of bringing the cells and the fragmented extracellular matrix components into contact in an aqueous medium before incubation. [Examples]

[0095] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0096] By heating 100 mg of porcine skin-derived collagen type I sponge fragments (manufactured by Nippon Ham Co., Ltd.) at 200°C for 24 hours, a collagen component in which at least a portion was cross-linked (cross-linked collagen component) was obtained. No significant external changes were observed in the collagen before and after heating at 200°C. 50 mg of the cross-linked collagen component was placed in a 15 mL tube, 5 mL of ultrapure water was added, and the cross-linked collagen component was defibrillated by homogenization for 6 minutes using a homogenizer (AS ONE VH-10).

[0097] Under conditions of 21°C, the collagen pellet was centrifuged at 10,000 rpm for 10 minutes. The supernatant was aspirated, and the collagen pellet was mixed with 5 mL of fresh ultrapure water to prepare a collagen solution. The tube containing the collagen solution was kept on ice and sonicated for 20 seconds at 100V using a sonicator (Sonics and Materials VC50). After removing the sonicator, the tube containing the collagen solution was cooled on ice for 10 seconds, and this process was repeated 100 times. After 100 sonication cycles, the collagen solution was filtered through a 40 μm pore size filter to obtain a dispersion containing defibrated collagen components (CMF). The dispersion was freeze-dried by a conventional method to obtain defibrated collagen components (CMF) as a dried product. The average length of the CMF was 14.8 ± 8.2 μm (N=20). For use, the fragmented collagen was dispersed in a serum-containing culture medium (DMEM) to a concentration of 10 mg / mL.

[0098] The cells, reagents, and preparation methods used in the creation of the three-dimensional tissue are as follows. (Cells and collagen) FBS (Thermo Fisher, Gibco) • Subcutaneous fat-derived stem cells (ADSC, derived from cattle, collected by conventional methods from beef intended for meat consumption) • Erucic acid (Sigma-Ace #E3385) • Elaidic acid (Sigma-America #E4637) • Oleic acid (Sigma-Ace #O1008) • Palmitoleic acid (Sigma-Agent #P9417) • Myristoleic acid (Sigma-America #M3525) • Phytanic acid (Sigma-America #P4060, or Laroda Fine Chemicals 11-1600) • Pristanic acid (Sigma-America #P6617, or Laroda Fine Chemicals 11-1500) • Bovine plasma-derived fibrinogen type IS (Sigma-Ace #F8630)

[0099] (Culture medium and various solutions) • DMEM medium (high glucose, Nacalai Tesque): Hereafter, when simply referred to as DMEM, it refers to a medium containing 10% FBS. • 50 mg / mL fibrinogen stock solution: Weigh 50 mg of fibrinogen into an Eppendorf tube and immediately add 1 mL of DMEM (0% FBS, 1% antibiotic). Mix by hand by shaking the tube, then place in a 37°C water bath for 3-5 minutes, filter through a 0.2 μm pore filter, and dispense an equal volume into an Eppendorf tube before use.

[0100] <Manufacturing of three-dimensional tissues> Manufacturing Example 1 The obtained defibrated collagen component was 1.2% by weight (final concentration), fibrinogen at 6 mg / mL (final concentration), thrombin at 3 U / mL (final concentration), and 5 × 10⁻¹⁰ 6 Two μL of FBS-free DMEM containing bovine subcutaneous adipose-derived stem cells at a concentration of cells / mL was seeded onto a 96-well microplate (IWAKI Corporation) and incubated for 15 minutes. After 15 minutes, 200 μL of DMEM medium was added.

[0101] [Table 1]

[0102] Two days later, the culture medium was changed with each medium shown in Table 1 above, and thereafter the medium was changed every two days until day 5 (7 days from seeding), day 9 (11 days from seeding), and day 13 (15 days from seeding) to obtain three-dimensional tissues. Each obtained three-dimensional tissue was lipid-stained with Nile Red, and the nucleus was stained with Hoechst stain. Fluorescence observation was performed using a confocal quantitative image cytometer CQ1 (YOKOGAWA), and the total fluorescence intensity of lipid staining (Nile Red) was standardized by nuclear (Hoechst) quantification using image analysis software (ImageJ, NIH), then standardized under undifferentiated conditions (condition with only DMEM set to 1), and the fluorescence intensity of lipid staining (Nile Red) was calculated relative to the fluorescence intensity of nuclear staining (Hoechst) (2-3 samples were used for each condition).

[0103] The results are shown in Figure 1. In particular, it was shown that differentiation of adipose-derived stem cells was promoted in the three-dimensional tissue after 5 days of culture by culturing in DMEM medium containing free fatty acids.

[0104] Manufacturing Example 2 1.2% by weight (final concentration) of defibrated collagen, 6 mg / mL (final concentration) of fibrinogen, 3 U / mL (final concentration) of thrombin, and 5 × 10 6 Two μL of FBS-free DMEM containing bovine subcutaneous adipose-derived stem cells at a concentration of cells / mL was seeded onto a 96-well plate (IWAKI Corporation) and incubated for 15 minutes. Then, 200 μL of the culture medium shown in Table 2 below was added. Two days later, the culture medium was changed with each medium shown in Table 2 below, and the medium was changed every two days until day 7 (day 9 after seeding) to produce three-dimensional tissues.

[0105] [Table 2]

[0106] Figure 2 shows the results of comparing the fluorescence intensity of lipid staining (Nile Red) with that of nuclear staining (Hoechst) after staining each three-dimensional tissue at day 7 of differentiation (day 9 from seeding) in the same manner as in Production Example 1. Compared with conventionally used cell media (No. 1-4), the differentiation of adipose-derived stem cells was shown to be promoted in three-dimensional tissue cultured in DMEM medium (No. 5) containing seven types of free fatty acids.

[0107] Manufacturing Example 3 1.2% by weight (final concentration) of defibrated collagen, 6 mg / mL (final concentration) of fibrinogen, 3 U / mL (final concentration) of thrombin, and 5 × 10 6 Two μL of FBS-free DMEM containing subcutaneous adipose-derived stem cells at a concentration of cells / mL was seeded onto a 96-well plate (IWAKI Corporation) and incubated for 15 minutes. Then, 200 μL of DMEM medium was added, and the cells were cultured for 48 hours. Subsequently, the culture medium was changed with 200 μL of DMEM medium containing 50 μM each of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid, and 0 μM, 1 μM, 5 μM, or 10 μM of the ALK5 inhibitor (2-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]-1,5-naphthirizine, Cayman Chemical No. 14794). The medium was changed every two days, and the cells were cultured until day 7 of differentiation (day 9 from seeding) to obtain a three-dimensional tissue.

[0108] Each obtained three-dimensional tissue was lipid-stained with Nile Red, and the nucleus was stained with Hoechst stain. Similar to Production Example 1, the fluorescence intensity of lipid staining (Nile Red) was calculated relative to the fluorescence intensity of nuclear staining (Hoechst), and the rate of increase in fluorescence intensity was calculated relative to three-dimensional tissue cultured in DMEM medium without ALK5 inhibitors (three samples were used for each condition).

[0109] Figure 3 shows images of each three-dimensional tissue on day 7 of differentiation (day 9 of culture) observed using a confocal quantitative image cytometer CQ (YOKOGAWA) for fluorescence detection. Figure 4 shows a comparison of the rate of increase in fluorescence intensity in each three-dimensional tissue.

[0110] Compared to three-dimensional tissues prepared in DMEM medium without the ALK5 inhibitor, three-dimensional tissues prepared in DMEM medium containing the ALK5 inhibitor showed more advanced adipogenesis (Figure 3), and a corresponding increase in fluorescence intensity was observed (Figure 4). In particular, a more than threefold increase in fluorescence intensity was observed when DMEM medium containing 5 μM or more of the ALK5 inhibitor was used (Figure 4). This indicates that the addition of the ALK5 inhibitor promotes the differentiation of adipose-derived stem cells into mature adipocytes.

[0111] Manufacturing Example 4 Fibrin-disintegrated collagen component 1.2% by weight (final concentration), 6 mg / mL (final concentration) fibrinogen, 3 U / mL (final concentration) thrombin, 5 × 10 6 Two μL of FBS-free DMEM containing subcutaneous adipose-derived stem cells at a concentration of cells / mL was seeded onto a 96-well plate (IWAKI Corporation) and incubated for 15 minutes. Then, 200 μL of DMEM medium was added and the cells were cultured for 48 hours. Subsequently, the culture medium was changed with 200 μL of DMEM medium containing 50 μM each of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid, and 5 μM of the ALK5 inhibitor (2-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]-1,5-naphthirizine, Cayman Chemical No. 14794). The medium was changed every two days and the culture was continued to obtain three-dimensional tissues.

[0112] Figure 5 shows images of three-dimensional tissues at 3 days of differentiation (5 days after seeding), 7 days of differentiation (9 days after seeding), and 14 days of differentiation (16 days after seeding), stained with Nile Red for lipids, stained with Hoechst stain for the nuclei, and observed for fluorescence using a confocal quantitative image cytometer CQ (YOKOGAWA). Figure 6 shows the percentage increase in fluorescence intensity in each three-dimensional tissue compared to three-dimensional tissues cultured for the same period in DMEM medium without an ALK5 inhibitor, similar to Production Example 3.

[0113] Adipogenesis was observed in all three-dimensional tissues at days 3, 7, and 14 of differentiation (Figure 5), and an increase in fluorescence intensity was observed (Figure 6). Adipogenesis was particularly advanced in the three-dimensional tissues at days 7 and 14 of culture (Figure 5), and the increase in fluorescence intensity was also significant (Figure 6). There was not much difference in the rate of increase in fluorescence intensity between the three-dimensional tissues at days 7 and 14 of culture (Figure 6).

[0114] Manufacturing Example 5 1.2% by weight (final concentration) of defibrated collagen, 6 mg / mL (final concentration) of fibrinogen, 3 U / mL (final concentration) of thrombin, and 5 × 10 6 Three-dimensional tissues were obtained by seeding 2 μL of FBS-free DMEM containing bovine subcutaneous adipose-derived stem cells at a concentration of cells / mL into a 96-well microplate, incubating for 15 minutes, and then adding DMEM medium and culturing for 48 hours.

[0115] The cells were cultured in 200 μL of DMEM medium (1) to (3) below, with the medium being changed every two days. (1) DMEM medium (1×FFA) containing 50 μM each of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid; (2) DMEM medium (2×FFA) containing 100 μM each of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid; (3) DMEM medium (3×FFA) containing 150 μM each of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid.

[0116] Figure 7 shows the results of comparing the fluorescence intensity of lipid staining (Nile Red) with the fluorescence intensity of nuclear staining (Hoechst) in the same manner as in Production Example 1, for three-dimensional tissues cultured in DMEM medium from (1) to (3) at 3 days of differentiation (5 days after seeding), 7 days of differentiation (9 days after seeding), and 14 days of differentiation (16 days after seeding). Three-dimensional tissues cultured in DMEM medium containing erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid, and especially three-dimensional tissues cultured in medium containing 100 μM of each of the above fatty acids, showed a higher degree of adipocyte maturation and promoted differentiation of adipose-derived stem cells.

[0117] Similarly, three-dimensional tissues from days 3, 7, and 14 of differentiation were stained with Nile Red and Hoechst, and fluorescence images obtained using a confocal quantitative image cytometer CQ (YOKOGAWA) are shown in Figures 8-10. Figure 8 shows the three-dimensional tissue from day 3 of differentiation, Figure 9 shows the tissue from day 7 of differentiation, and Figure 10 shows the tissue from day 14 of differentiation.

[0118] Adipogenesis was confirmed in all three-dimensional tissues at days 3, 7, and 14 of differentiation (Figures 8-10). Even in culture after tissue formation, the differentiation-promoting effects of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid on adipose-derived stem cell differentiation were observed.

[0119] Manufacturing Example 6 1.2% by weight (final concentration) of defibrated collagen, 6 mg / mL (final concentration) of fibrinogen, 3 U / mL (final concentration) of thrombin, and 5 × 10 6 Three-dimensional tissues were obtained by seeding 2 μL of FBS-free DMEM containing bovine subcutaneous adipose-derived stem cells at a concentration of cells / mL into a 96-well microplate, incubating for 15 minutes, and then adding DMEM medium and culturing for 48 hours.

[0120] The cells were cultured in 200 μL of the following DMEM medium (1)~(2), with the medium being changed every two days. (1) DMEM medium (7 FFA) containing 100 μM each of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid, (2) DMEM medium (6 FFA) containing 100 μM each of elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid.

[0121] Figure 11 shows the results of comparing the fluorescence intensity of lipid staining (Nile Red) with that of nuclear staining (Hoechst) after 7 days of differentiation (9 days after seeding) in DMEM medium (1) and (2), as in the same manner as in Production Example 1. The maturity of adipocytes was equivalent in the three-dimensional tissue cultures cultured in medium containing 6 types of fatty acids and the three-dimensional tissue cultured in medium containing 7 types of fatty acids, indicating that the differentiation of adipose-derived stem cells was similarly promoted.

Claims

[Claim 1] A method for producing a three-dimensional tissue containing mature adipocytes, comprising incubating cells containing at least adipose-derived stem cells in the presence of one or more fatty acids selected from the group consisting of erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid.

Images