Method for producing tissue and method for promoting differentiation of adipose-derived stem cells

Abstract

Disclosed is a method for producing a tissue body including vascular cells, the method comprising incubating cells at least including fat-derived stem cells in the presence of equine serum.

Description

[Technical Field]

[0001] The present invention relates to a method for producing 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 vascular cells are expected to be used as a substitute for experimental animals and as transplant material from the standpoint of maintaining the three-dimensional tissue and ensuring engraftment when transplanted. Therefore, a method to promote the differentiation of adipose-derived stem cells into vascular cells is desired when manufacturing three-dimensional tissues containing vascular cells.

[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 vascular cells in the production of tissues containing vascular cells. [Means for solving the problem]

[0006] As a result of diligent research, the inventors discovered that incubating adipose-derived stem cells in the presence of horse serum promotes the differentiation of adipose-derived stem cells into vascular cells, thus completing the present invention.

[0007] In other words, the present invention includes, for example, the following inventions. [1] A method for producing a tissue containing vascular cells, comprising incubating cells containing at least adipose-derived stem cells in the presence of horse serum. [2] The method for producing a tissue containing vascular cells having a vascular network, as described in [1]. [3] A method for producing cells according to [1] or [2], wherein the cells containing at least adipose-derived stem cells do not contain vascular cells. [4] The method for producing the product according to any one of [1] to [3], wherein incubation in the presence of the above-mentioned horse serum is performed by incubation in a culture medium containing the above-mentioned horse serum. [5] The manufacturing method according to any one of [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 [1] to [5], wherein incubation is performed for 144 hours or more. [7] A production method according to any one of [1] to [6], comprising incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of equine serum. [8] The production method according to [7], wherein in the tissue mass containing the vascular cells, the fragmented extracellular matrix components are arranged in the gaps between the cells. [9] The production method according to [7] or [8], wherein the fragmented extracellular matrix components are fragmented collagen components.

[10] Before incubation, it includes a step of contacting the cells and the fragmented extracellular matrix components in an aqueous medium. Incubating cells containing at least adipose-derived stem cells in the presence of equine serum is a step of incubating the cells contacted with the fragmented extracellular matrix components in the presence of equine serum. The tissue mass containing the vascular cells is a three-dimensional tissue mass containing vascular cells. The production method according to any one of [7] to [9].

[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 equine serum.

[12] The method according to

[11] , wherein incubating in the presence of the equine serum is incubating in a medium containing the equine serum.

[13] The method according to

[11] or

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

[14] The method according to any one of

[11] to

[13] , wherein the incubation is performed for 144 hours or more.

[15] A method according to any one of

[11] to

[14] , comprising incubating cells containing at least adipose-derived stem cells and fragmented extracellular matrix components in the presence of equine serum.

[16] The method according to

[15] , wherein the fragmented extracellular matrix component is a fragmented collagen component.

Advantages of the Invention

[0008] According to the present invention, the differentiation of adipose-derived stem cells into vascular cells is promoted. By promoting the differentiation into vascular cells, it becomes possible to produce a tissue mass containing vascular cells in a short time.

Brief Description of the Drawings

[0009] [Figure 1] FIG. 1 shows the results of CD31 immunostaining of the tissue mass prepared in Production Example 1. [Figure 2] FIG. 2 shows the results of CD31 immunostaining and counterstaining with hematoxylin of the tissue mass prepared in Production Example 2. [Figure 3] FIG. 3 shows the results of CD31 immunostaining and counterstaining with hematoxylin of the tissue mass prepared in Production Example 3. The white dots indicate nuclei (hematoxylin staining). [Figure 4] FIG. 4 shows the results of CD31 immunostaining and counterstaining with hematoxylin of the tissue mass prepared in Production Example 4. The white dots indicate nuclei (hematoxylin staining). [Figure 5] FIG. 5 shows the results of CD31 immunostaining and counterstaining with hematoxylin of the tissue mass prepared in Production Example 5. The white dots indicate nuclei (hematoxylin staining). [Figure 6] FIG. 6 shows the results of quantification of CD31 immunostaining of the tissue mass prepared in Production Example 6. [Figure 7] FIG. 7 shows the results of quantification of CD31 immunostaining of the tissue mass prepared in Production Example 6.

Modes for Carrying Out the Invention

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

[0011] As one embodiment, the present invention provides a method for producing a tissue body containing vascular cells, comprising incubating cells containing at least adipose-derived stem cells in the presence of horse serum.

[0012] In this specification, "tissue" means a tissue containing cells artificially created by cell culture. A "tissue" may be a "two-dimensional tissue" in which cells are arranged two-dimensionally, or a "three-dimensional tissue" which is an aggregate of cells (a clump of cells) in which cells are arranged three-dimensionally. A two-dimensional tissue is a tissue in which cells are cultured in a planar shape, for example, in which the cells exist in a shape that is stretched out in a planar shape on a substrate. If a 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 a three-dimensional tissue, and examples include sheet-like, spherical, nearly spherical, ellipsoidal, nearly ellipsoidal, hemispherical, nearly hemispherical, semicircular, nearly semicircular, rectangular parallelepiped, nearly rectangular parallelepiped, etc. Here, living tissue includes sweat glands, lymphatic vessels, sebaceous glands, etc., and its structure is more complex than that of a three-dimensional tissue. Therefore, three-dimensional tissues and living tissues can be easily distinguished.

[0013] In this specification, "cells" are not particularly limited, but may be cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cattle, 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 lines.

[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. Examples of adipose-derived stem cells (ADSCs) include human adipose-derived stem cells and bovine adipose-derived 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 tissue body containing mature adipocytes produced by the method of this embodiment (e.g., a three-dimensional tissue body) is ultimately used to represent the tissue of a specific part of a living organism, it is preferable to use stem cells derived from the tissue corresponding to that part. Furthermore, the adipose-derived stem cells may be, for example, stem cells derived from cattle, horses, mice, rats, pigs, and humans. 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 vascular cells, differentiation into vascular cells 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 include cells other than adipose-derived stem cells. Examples of cells other than adipose-derived stem cells include vascular endothelial cells, adipocytes, 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). 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. 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, so it is preferable that the cells containing at least adipose-derived stem cells, i.e., the cells before incubation, do not contain vascular cells.

[0017] In the cells before incubation, it is preferable that 90% or more of the total number of cells are adipose-derived stem cells, and it is more preferable that all of them are adipose-derived stem cells.

[0018] The cells may further include vascular endothelial cells. In this specification, “vascular endothelial cells” means flattened cells that make up the surface of the vascular lumen. Examples of vascular endothelial cells include human umbilical vein-derived vascular endothelial cells (HUVECs).

[0019] 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.

[0020] The tissue includes at least vascular cells. Vascular cells include, for example, vascular endothelial cells. In this specification, “vascular endothelial cells” means the flattened cells that make up the surface of the vascular lumen.

[0021] The percentage of vascular cells in a tissue 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 tissue, and may be 95% or less, 90% or less, 80% or less, or 75% or less.

[0022] If the tissue contains vascular endothelial cells, the vascular endothelial cell 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 tissue, and may be 95% or less, 90% or less, 80% or less, or 75% or less.

[0023] The tissue may further contain cells other than vascular cells. Examples of cells other than vascular 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 vascular cells, the effects of the present invention become more pronounced. Therefore, it is preferable that the tissue after incubation, as described later, contains 10% or less adipose-derived stem cells relative to the total number of cells in the tissue, more preferably 5% or less, and even more preferably no adipose-derived stem cells at all.

[0024] The tissue may have a vascular network between its cells. The presence of a vascular network between cells is expected to allow for long-term maintenance of the tissue and to facilitate engraftment when transplanted into mammals or other organisms.

[0025] "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 the 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 the 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 of the distance between blood vessel branches in the tissue is 80% to 150%, 85% to 130%, and 90% to 120% of the average length of the distance between blood vessel branches in living tissue, it may be judged to be similar to the distance between blood vessel branches 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. When the tissue contains adipocytes, it is preferable that the tissue has a vascular network between the 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 tissue according to this embodiment is 20 μm to 180 μm, or 100 μm to 180 μm, it may be determined that the tissue has adipocytes similar to those in living tissue. When comparing living tissue and the tissue, the comparison is made under the same conditions (for example, per a certain volume, per a certain area in the case of image analysis, per a certain sample, etc.).

[0026] When the tissue is a three-dimensional tissue, its thickness 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.

[0027] 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.

[0028] 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.

[0029] The method of this embodiment includes incubating cells containing at least adipose-derived stem cells in the presence of horse serum. Incubation in the presence of horse serum promotes the differentiation of adipose-derived stem cells into vascular cells.

[0030] In this embodiment, the method only requires that differentiation be promoted by contact of at least some adipose-derived stem cells with horse serum. Therefore, the method of tissue production is not particularly limited and may be three-dimensional or two-dimensional culture. In this embodiment, a tissue containing vascular cells can ultimately be produced by promoting differentiation while the adipose-derived stem cells are included in the tissue, or by promoting differentiation in a situation where a tissue is formed so that adipose-derived stem cells are included in the tissue. Therefore, incubating cells containing at least adipose-derived stem cells in the presence of horse serum may involve incubating only the cells in the presence of horse serum, incubating a mixture of cells and extracellular matrix components in the presence of horse serum, or incubating a tissue containing cells and extracellular matrix components in the presence of horse serum after a tissue has been formed.

[0031] The production of tissues by two-dimensional culture can be carried out by known methods for culturing cells in a planar manner. Examples include methods comprising adding cells and culture medium to a substrate and culturing the cells, and methods comprising coating a substrate with a gel, adding cells and culture medium to the coating and culturing the cells. As the gel used to coat the substrate, the above-mentioned fibrin gel, hydrogel, Matrigel, collagen gel, and gelatin gel can be used.

[0032] While tissues can be produced by known methods through three-dimensional culture, preferred examples for producing three-dimensional tissues using fragmented extracellular matrix components will be described later.

[0033] Horse serum (HS) can be prepared by conventional methods, but commercially available serum may also be used. Examples of commercially available horse serum include S0900 (BioWest), SH30074.02 (Cytiva), and 16050130 (ThermoFisher).

[0034] Incubating in the presence of horse serum may, for example, mean incubating (cultivating) cells containing at least adipose-derived stem cells in a culture medium containing horse serum.

[0035] There are no particular restrictions on the culture medium, and a suitable medium can be selected depending on the type of cells to be cultured. The medium may be a solid medium or a liquid medium, but a liquid medium is preferred. Examples of media include Eagle's MEM medium, DMEM, F12K medium, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. In addition, a gel-like liquid medium such as fibrin gel, hydrogel, Matrigel, collagen gel, and gelatin gel may be used. Cells may be added to a gel-like liquid medium, or a liquid medium containing cells may be gelled before use. The medium may contain serum other than horse serum, or it may not contain serum other than horse serum. The medium may be a mixed medium obtained by mixing two types of media.

[0036] Horse serum contains adipose-derived stem cells 1 x 10 4 The amount per cell may be 20-400 μL, 20-240 μL, or 160-240 μL.

[0037] When cells containing at least adipose-derived stem cells are incubated in a culture medium containing horse serum, the horse serum content in the culture medium may be, for example, 1% to 20%, 3% to 17%, 4% to 15%, or 5% to 12%, or 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more, or 20% or less, 18% or less, 16% or less, 14% or less, or 12% or less.

[0038] 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 tissue. For example, the cell density in the culture medium can be 1 to 10 8 It may be cells / mL, 10 3~10 7 It may be cells / mL, 10 5 ~10 6 It may also be cells / mL.

[0039] 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, or 96 hours to 288 hours. The incubation time in the presence of horse serum may be 24 hours to 336 hours, 72 hours to 336 hours, or 96 hours to 288 hours. In addition, the incubation time in the presence of horse serum may be 72 hours or more, 84 hours or more, 96 hours or more, 108 hours or more, 120 hours or more, 132 hours or more, or 144 hours or more, and may be 336 hours or less, 312 hours or less, 288 hours or less, 264 hours or less, 240 hours or less, or 226 hours or less.

[0040] 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.

[0041] 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 horse serum. 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 the cells may be homogeneous or heterogeneous.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Examples of proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

[0046] 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.

[0047] 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.

[0048] "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.

[0049] 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 in an aqueous medium such as physiological saline. 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] The degree of crosslinking may be calculated by quantifying the carboxyl groups. For example, in the case of water-insoluble extracellular matrix components, quantification may be performed by the TBO (toluidine blue O) method. The degree of crosslinking obtained by the TBO method may be within the range described above.

[0063] The extracellular matrix component content in the tissue (e.g., a 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.

[0064] Here, "extracellular matrix components in a tissue" refers to the extracellular matrix components that make up the tissue, and may originate from endogenous extracellular matrix components or from exogenous extracellular matrix components.

[0065] "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.

[0066] "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.

[0067] 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.

[0068] 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.

[0069] In other words, when a tissue contains endogenous extracellular matrix components and fragmented extracellular matrix components, the extracellular matrix component content of the 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 tissue and the mass of the decellularized tissue.

[0070] 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.

[0071] More specifically, the following methods can be used to quantify the amount of collagen.

[0072] (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.

[0073] (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.

[0074] (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.

[0075] The collagen component in a 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 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 area of ​​collagen component to the entire tissue using macroscopic observation, various microscopes, and image analysis software. When defining by area ratio, there are no limitations on which cross-section or surface of the tissue is used to define the area ratio; however, if the tissue is a three-dimensional tissue, such as a sphere, it may be defined by a cross-sectional view passing through its approximate center.

[0076] For example, when defining the collagen component in a 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 tissue. The area ratio of the collagen component constituting the tissue refers to the area ratio of endogenous collagen component and exogenous collagen component combined. The area ratio of the collagen component can be calculated, for example, by staining the obtained tissue with Masson's trichrome and taking the ratio of the area of ​​the blue-stained collagen component to the total area of ​​a cross-section passing through approximately the center of the tissue.

[0077] The tissue material preferably retains 70% or more after trypsin treatment at a trypsin 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 tissue material is less susceptible to enzymatic degradation during or after culture and is stable. The above retention rate can be calculated, for example, from the mass of the tissue material before and after trypsin treatment.

[0078] The above 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 tissues are less susceptible to enzymatic degradation during or after culture and are stable.

[0079] Incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of horse serum means, for example, incubating (culturing) the cells in a medium containing horse serum and fragmented extracellular matrix components. In this case, for example, a medium containing fragmented extracellular matrix components and horse serum beforehand may be used, horse serum may be added to a medium containing fragmented extracellular matrix components, or fragmented extracellular matrix components may be added to a medium containing horse serum.

[0080] 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 horse serum may be included 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 arranged 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 in three dimensions. Therefore, if the process further includes a step of contacting the cells with the fragmented extracellular matrix components in an aqueous medium, incubating cells containing at least adipose-derived stem cells in the presence of horse serum may be equivalent to incubating the cells that have come into contact with the fragmented extracellular matrix components in the presence of horse serum. In this case, the tissue containing vascular cells produced is a three-dimensional tissue containing vascular cells.

[0081] 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. Examples of aqueous mediums include, but are not limited to, salines such as phosphate-buffered saline (PBS), Dulbecco's Modified Eagle medium (DMEM), F12K medium, and vascular endothelial cell medium (EGM2).

[0082] 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, F12K medium, 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] One embodiment of a method for producing a three-dimensional tissue may include mixing fibrinogen and thrombin simultaneously or separately in a contact step, or after the contact step and before the culture step. 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.

[0089] 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.

[0090] 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.

[0091] As one embodiment, the present invention also provides a method for promoting the differentiation of adipose-derived stem cells, which includes incubating cells containing at least adipose-derived stem cells in the presence of horse serum. As described above, the differentiation of adipose-derived stem cells into vascular cells can be promoted by incubating adipose-derived stem cells in the presence of horse serum. The method of this embodiment can also be used in the production of tissues, which may be produced by three-dimensional culture or two-dimensional culture.

[0092] The specific aspects of the method of this embodiment, including 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.

[0093] Furthermore, the method of this embodiment may also be a method for promoting the differentiation of adipose-derived stem cells in a method for manufacturing a three-dimensional tissue.

[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 as described above. [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] The cells, reagents, and preparation methods used in the preparation of the tissue samples are as follows: (Cells and collagen) • Subcutaneous fat-derived stem cells (ADSC, derived from cattle, collected by conventional methods from beef intended for meat consumption) • Bovine plasma-derived fibrinogen type IS (Sigma-Ace #F8630) • Horse serum (HS, ThermoFisher #16050130) • Fetal bovine serum (FBS, ThermoFisher #26140) • Calf serum (GE Healthcare #SH30118) • Human serum (Lonza #4W-820)

[0097] (Culture medium and various solutions) • DMEM medium (high glucose, Nacalai Tesque) • F12K medium (ThermoFisher #21127022) • 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 equal volumes into Eppendorf tubes before use.

[0098] Manufacturing Example 1 Tissue samples were prepared using two-dimensional culture as follows. A 48-well plate (manufactured by IWAKI Corporation) was prepared. 5000 bovine subcutaneous adipose-derived stem cells were seeded in each well, and the cells were cultured for 7 days in 1 mL each of DMEM medium containing 10% fetal bovine serum, F12K medium containing 10% fetal bovine serum, DMEM medium containing 10% horse serum, and F12K medium containing 10% horse serum.

[0099] Each of the obtained tissue bodies was subjected to CD31 immunostaining, and the photograph obtained by fluorescence observation using a confocal quantitative image cytometer CQ (YOKOGAWA) is shown in Fig. 1. "×4" and "×20" indicate that the photographs were observed at magnifications of 4 times (left) and 20 times (right) in each medium, respectively.

[0100] In the tissue bodies prepared in the medium containing fetal bovine serum, endothelial formation was not confirmed. On the other hand, in the tissue bodies prepared in the FK12 medium containing equine serum, endothelial formation was progressing, and it was shown that a vascular network was constructed. No difference due to the difference between the DMEM medium and the F12K medium was recognized. It was shown that the addition of equine serum promoted the differentiation of adipose-derived stem cells into vascular cells.

[0101] Production Example 2 Tissue bodies were prepared by two-dimensional culture as follows. A 48-well plate was prepared in the same manner as in Production Example 1. In 200 μL of an F12K medium or a DMEM medium containing 10% equine serum, 5×10 5 cells / mL or 1×10 6 cells / mL of bovine subcutaneous adipose-derived stem cells were cultured for 7 days, seeded on each well, and each tissue body obtained 48 hours later was evaluated by CD31 immunostaining and counterstaining with Hoechst.

[0102] The photograph obtained by fluorescence observation using a confocal quantitative image cytometer CQ (YOKOGAWA) is shown in Fig. 2. What is observed as white dots on the right is the nucleus.

[0103] In both cases where the cell number was 5×10 5 cells and 1×10 6 cells, it was confirmed that a vascular network was constructed between cells due to endothelial formation. No difference due to the difference between the DMEM medium and the F12K medium was recognized. It was shown that the addition of equine serum promoted the differentiation of adipose-derived stem cells into vascular cells regardless of the cell number.

[0104] Production Example 3 Tissue samples were prepared using two-dimensional culture as follows. Bovine subcutaneous adipose-derived stem cells were seeded at a rate of 5000 cells / well in 24-well or 48-well plates (manufactured by IWAKI Corporation), and cultured for 7 days in 1 mL of DMEM medium containing 1%, 5%, and 10% horse serum, respectively, to obtain tissue samples.

[0105] Each obtained tissue sample was evaluated by CD31 immunostaining and counterstaining with Hoechst. Fluorescence images obtained using a confocal quantitative image cytometer CQ (YOKOGAWA) are shown in Figure 3. For each horse serum content, the cell concentration was 2600 (cells / cm³). 2 The result obtained using a 24-well plate was 5200 (cells / cm²). 2 The result shown is that using a 48-well plate.

[0106] Compared to the case using 1% horse serum, endothelial formation was more advanced when 5% horse serum was used, and even more advanced when 10% horse serum was used. It was shown that the more horse serum added, the more the differentiation of adipose-derived stem cells into vascular cells was promoted.

[0107] Manufacturing Example 4 Tissue samples were prepared using two-dimensional culture as follows. Bovine subcutaneous adipose-derived stem cells were seeded at a rate of 5000 cells / well in 24-well or 48-well plates (manufactured by IWAKI Corporation), and cultured for 7 days in 1 mL of DMEM medium containing 10% fetal bovine serum, fetal bovine serum, equine serum, and human serum, respectively, to obtain tissue samples.

[0108] Each obtained tissue sample was evaluated by CD31 immunostaining and counterstaining with Hoechst. Fluorescence images obtained using a confocal quantitative image cytometer CQ (YOKOGAWA) are shown in Figure 4. In each culture medium, the cell concentration was 2600 (cells / cm³). 2 The result obtained using a 24-well plate was 5200 (cells / cm²).2 The result shown is that using a 48-well plate.

[0109] In tissues prepared using horse serum, endothelial formation was observed to have created a vascular network between cells. However, in tissues prepared using fetal bovine serum, fetal bovine serum, and human serum, such advanced endothelial formation was not observed. Compared to cases using other serums, endothelial formation was clearly more advanced when using horse serum. This indicates that the addition of horse serum promotes the differentiation of adipose-derived stem cells into vascular cells.

[0110] Manufacturing Example 5 Three-dimensional tissue samples were prepared using three-dimensional culture as described below. 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).

[0111] 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).

[0112] After dispersing fragmented collagen in culture medium (F12K) to a concentration of 10 mg / mL, fibrinogen and cells were mixed together, resulting in a mixture of defibrated collagen components (final concentration 1.2 wt%), fibrinogen (final concentration 6 mg / mL), and bovine subcutaneous fat-derived stem cells (final concentration 2 × 10⁻¹⁴). 6 A mixture of cells / mL was obtained, and 30 μL of this mixture was seeded into a 24-well plate (IWAKI Corporation). 2 mL of culture medium containing 10% horse serum was added to the seeded mixture and cultured. The culture medium was changed every two days and cultured until day 10 to obtain a three-dimensional tissue.

[0113] Each obtained three-dimensional tissue was immunostained with CD31, and the nuclei were stained with Hoechst stain. Fluorescence images obtained using a confocal quantitative image cytometer CQ (YOKOGAWA) are shown in Figure 5. The image on the left shows only CD31 immunostaining, while the image on the right shows both CD31 immunostaining and Hoechst staining. The white dots on the right represent the nuclei.

[0114] In three-dimensional tissues prepared in FK12 medium containing 10% horse serum, the formation of a vascular network between cells through endothelial formation was confirmed. The addition of horse serum was shown to promote the differentiation of adipose-derived stem cells into vascular cells.

[0115] Manufacturing Example 6 Tissues were prepared using two-dimensional culture as described below, and the culture of bovine subcutaneous adipose-derived stem cells and their differentiation into bovine endothelial cells were observed.

[0116] A 48-well plate (manufactured by IWAKI Corporation) was prepared. 5000 first-passage bovine subcutaneous adipose-derived stem cells (1st passage) pre-cultured in DMEM medium were seeded into each well, and the cells were cultured for 7 days using the following eight different culture media, changing the medium every 2-3 days. (1) DMEM medium containing 1% horse serum (2) DMEM medium containing 5% horse serum (3) DMEM medium containing 10% horse serum (4) DMEM medium containing 10% fetal bovine serum (5) DMEM medium containing 10% human serum (6) DMEM medium containing 10% calf serum (7) F12K medium containing 10% horse serum (8) F12K medium containing 10% fetal bovine serum Note that all of the culture media (1) to (8) contain a 1% mixed solution of antimicrobial and antifungal agents.

[0117] Each obtained tissue sample was quantified using CD31 immunostaining (n=3), and the results of a comparison under different serum conditions in DMEM medium are shown in Figure 6 (one-way unpaired ANOVA with a Tukey's HSD post-test). The addition of horse serum, particularly 5% and 10% horse serum, was shown to promote the differentiation of adipose-derived stem cells into endothelial cells.

[0118] Furthermore, each obtained tissue sample was subjected to CD31 immunostaining quantification (n=3), and the results using 10% horse serum and 10% fetal bovine serum in different base media are shown in Figure 7 (two-ways unpaired ANOVA with a Sidak post-test). In tissue samples prepared in FK12 medium and DMEM medium containing 10% horse serum, differentiation of adipose-derived stem cells into endothelial cells was shown to be accelerated compared to tissue samples prepared in FK12 medium and DMEM medium containing 10% fetal bovine serum. Here again, no significant difference was observed between DMEM medium and F12K medium.

Claims

1. This includes incubating cells containing at least adipose-derived stem cells in the presence of horse serum. The incubation process includes bringing at least some of the adipose-derived stem cells into contact with the horse serum to promote the differentiation of the adipose-derived stem cells into vascular cells. Incubation was performed for more than 120 hours. The adipose-derived stem cells include bovine adipose-derived stem cells. A method for producing a tissue body containing vascular cells, wherein at least the cells containing the adipose-derived stem cells do not contain vascular cells.

2. The manufacturing method according to claim 1, wherein the tissue body containing vascular cells has a vascular network.

3. The method for producing the product according to claim 1 or 2, wherein incubation in the presence of the horse serum is incubation in a culture medium containing the horse serum.

4. The manufacturing method according to any one of claims 1 to 3, wherein the adipose-derived stem cells are derived from a bovine.

5. The manufacturing method according to any one of claims 1 to 4, wherein incubation is performed for 144 hours or more.

6. A method for producing a cell according to any one of claims 1 to 5, comprising incubating cells containing at least adipose-derived stem cells together with fragmented extracellular matrix components in the presence of horse serum.

7. The manufacturing method according to claim 6, wherein in the tissue body including the vascular cells, the fragmented extracellular matrix components are arranged in the spaces between the cells.

8. The manufacturing method according to claim 6 or 7, wherein the fragmented extracellular matrix component is a fragmented collagen component.

9. The process includes a step of bringing the cells and the fragmented extracellular matrix components into contact in an aqueous medium before incubation. Incubating cells containing at least adipose-derived stem cells in the presence of horse serum is The step involves incubating the cells that have come into contact with the fragmented extracellular matrix components in the presence of horse serum. The aforementioned tissue containing vascular cells is a three-dimensional tissue containing vascular cells. The manufacturing method according to any one of claims 6 to 8.

10. This includes incubating cells containing at least adipose-derived stem cells in the presence of horse serum. The incubation process includes bringing at least some of the adipose-derived stem cells into contact with the horse serum to promote the differentiation of the adipose-derived stem cells into vascular cells. Incubation was performed for more than 120 hours. The adipose-derived stem cells include bovine adipose-derived stem cells. A method for promoting the differentiation of adipose-derived stem cells, wherein the cells containing at least the adipose-derived stem cells do not contain vascular cells.

11. The method according to claim 10, wherein incubation in the presence of the horse serum is incubation in a culture medium containing the horse serum.

12. The method according to claim 10 or 11, wherein the adipose-derived stem cells are derived from bovine.

13. The method according to any one of claims 10 to 12, wherein incubation is performed for 144 hours or more.

14. The method according to any one of claims 10 to 13, comprising incubating cells containing at least adipose-derived stem cells and fragmented extracellular matrix components in the presence of horse serum.

15. The method according to claim 14, wherein the fragmented extracellular matrix component is a fragmented collagen component.

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