Cell structure and method for producing a cell structure

Abstract

To provide a cell construct having an intercellular vascular network, and a production method for such a cell construct having an intercellular vascular network.SOLUTION: Disclosed is a cell construct that contains a fragmented extracellular matrix component and cells and that has an intercellular vascular network, where the cells include at least fat cells and vascular endothelial cells.SELECTED DRAWING: None

Description

【Technical Field】 【0001】 The present invention relates to a cell structure and a method for manufacturing the cell structure, and particularly to a cell structure having a blood vessel network between cells and a method for manufacturing a cell structure having a blood vessel network between cells. 【Background Art】 【0002】 As a method for producing a structure artificially mimicking a living tissue, for example, a method for producing a three-dimensional tissue body including arranging cells coated with a film containing collagen three-dimensionally to form a three-dimensional tissue body (Patent Document 1), a method for producing a three-dimensional cell tissue including mixing cells with a cationic substance and an extracellular matrix component to obtain a mixture, collecting cells from the obtained mixture, and forming a cell aggregate on a substrate (Patent Document 2), etc. are known. Further, the present inventors have proposed a method for producing a three-dimensional tissue body having a large size with a thickness of 1 mm or more with a relatively small number of cells by bringing cells into contact with fragmented exogenous collagen (Patent Document 3). Such three-dimensional tissue bodies are expected to be used as substitutes for experimental animals, transplantation materials, etc. 【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 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0004】 The above-described method for manufacturing three-dimensional tissues allows for the creation of thick three-dimensional tissues. However, no method was known for creating adipose tissue in which a vascular network is formed between cells, similar to that of biological tissue. 【0005】 Therefore, the present invention aims to provide a cell structure having a vascular network between cells and a method for producing a cell structure having a vascular network between cells. [Means for solving the problem] 【0006】 In other words, the present invention relates to, for example, the following inventions. [1] comprising fragmented extracellular matrix components and cells, A cellular structure having a vascular network between cells, The above-mentioned cells are a cellular structure that includes at least adipocytes and vascular endothelial cells. [2] The cellular structure described in [1], wherein the above-mentioned vascular network is formed between the above-mentioned adipocytes. [3] The cell structure according to [1] or [2], wherein the adipocytes include mature adipocytes. [4] A cell structure according to any one of [1] to [3], wherein the average length of the fragmented extracellular matrix components is between 100 nm and 400 μm. [5] A cell structure according to any one of [1] to [4], wherein the extracellular matrix component content in the cell structure is 0.01 to 90% by mass, based on the dry weight of the cell structure. [6] A cell structure according to any one of [1] to [5], wherein the fragmented extracellular matrix component includes collagen. [7] A cell structure according to any one of [1] to [6], further comprising fibrin. [8] A cell structure described in any of [1] to [6], intended for transplantation. [9] A contact step of bringing fragmented extracellular matrix components into contact with cells, wherein the cells (i) include at least adipocytes, stem cells and vascular endothelial cells, or (ii) include at least adipocytes and vascular endothelial cells, A culture process in which cells in contact with fragmented extracellular matrix are cultured. A method for producing a cell structure having a vascular network between cells, including

[10] The method according to [9], wherein the cells include adipocytes, adipose stem cells, and vascular endothelial cells.

[11] The method according to [9] or

[10] , wherein the adipocytes include mature adipocytes.

[12] The amount of the fragmented extracellular matrix component in the above contact step is 1.0 × 10 6 The method according to any one of [9] to

[11] , wherein the amount is 0.1 to 100 mg relative to the cells.

[13] The method according to any one of [9] to

[12] , wherein the ratio of stem cells to vascular endothelial cells in the above contact step is 100 / 1 to 1 / 100.

[14] The method according to any one of [9] to

[13] , wherein the fragmented extracellular matrix component includes collagen.

[15] The method according to any one of [9] to

[14] , further comprising adding fibrinogen during the contact step or after the contact step and before the culture step.

[16] A non-human model animal having a cellular structure as a transplant, as described in any of [1] to [8].

[17] A method for producing a non-human model animal, comprising transplanting a cell structure described in any of [1] to [8] into a non-human animal.

[18] A method for transplanting a cell structure having a vascular structure, comprising transplanting the cell structure described in any of [1] to [8] into an animal.

[19] comprising fragmented extracellular matrix components and cells, The cells have a network of blood vessels between them. A cellular structure that is aggregated in a clump without being attached to a support, The above-mentioned cells are a cellular structure that includes at least adipocytes and vascular endothelial cells.

[20] A cell structure described in

[19] , which is roughly spherical in shape. 〔21〕A contacting step of contacting fragmented extracellular matrix components with cells, wherein the cells (i) contain at least adipocytes, stem cells and vascular endothelial cells, or (ii) contain at least adipose stem cells and vascular endothelial cells, A culturing step of culturing the cells contacted with the fragmented extracellular matrix and comprising, The above culturing step comprises culturing the cells contacted with the fragmented extracellular matrix in a state where they do not adhere to a support, and is a method for producing a cell structure having a vascular network between cells. 〔22〕The method according to

[21] , wherein the culturing step comprises separating the cells contacted with the fragmented extracellular matrix from the support. 〔23〕A cell tissue comprising a plurality of cell structures according to

[19] or

[20] , wherein the vascular network is connected between the plurality of cell structures. 〔24〕A method for producing a cell tissue, comprising suspension-culturing a plurality of cell structures according to

[19] or

[20] . 〔25〕A method for producing a non-human model animal, comprising transplanting a plurality of cell structures according to

[19] or

[20] into a non-human animal. 〔26〕The method according to

[25] , comprising growing for 30 days or more after transplanting the cell structure into a non-human animal. 〔27〕The method according to

[26] , comprising growing for 90 days or more after transplanting the cell structure into a non-human animal. 【Advantages of the Invention】 【0007】 According to the present invention, a cell structure having a vascular network between cells can be easily produced. 【Brief Description of the Drawings】 【0008】 [Figure 1] It is a photograph showing the observation results of (a) biological tissue and (b) the cell structure of Test Example 2 by perilipin staining and CD31 staining. [Figure 2] It is a graph comparing the number of vascular branches in the cell structure of Test Example 2 with the number of vascular branches in biological tissue. [Figure 3] It is a photograph showing the observation results of the cell construct of Test Example 3 by perilipin staining and CD31 staining. [Figure 4] It is a photograph showing the observation results of the cell construct of Test Example 4 by CD31 staining. [Figure 5] It is a photograph showing the observation results of the cell construct of Test Example 5 by CD31 staining. [Figure 6] It is a photograph showing the observation results of the cell construct of Test Example 6 by perilipin staining and CD31 staining. [Figure 7] It is a diagram showing the outline of Test Example 7. The circles in the left droplet represent mature adipocytes, the white rhombuses represent ADSCs, and the gray short bars represent HUVECs, respectively. [Figure 8] It is a photograph showing the fluorescence observation results of the cell ball with a blood vessel network of Test Example 7 by Nile red staining and CD31 staining. The right photograph is a further enlarged view of one of the cell balls on the left. [Figure 9] It is a photograph showing the observation results of the cell ball with a blood vessel network by CD31 staining. [Figure 10] It is a graph showing the average diameter (n = 12 cell balls / quantity) after culturing for 7 days of the cell balls prepared by the method of Test Example 7. [Figure 11] It is a photograph (left, upper right are partial enlarged photographs) showing the fluorescence observation results of the cell tissue prepared by the method of Test Example 8 by Nile red staining and CD31 staining and a photograph (lower right) of the aggregated cell balls on the plate observed under bright field. [Figure 12] It is a photograph showing the fluorescence observation results of the tissue collected 30 days after transplantation of Test Example 9 by perilipin staining and DAPI staining. A: SFT represents the tissue collected from the site where adipose tissue obtained by liposuction from a human thigh (biological tissue) was transplanted, C: 3DVFT represents the tissue collected from the site where the cell ball of (1) of Test Example 9 was transplanted. The upper part of Fig. 12 shows the results observed under bright field, the middle part shows the results by perilipin staining, and the lower part shows the results of DAPI staining. [Figure 13]This photograph shows the fluorescence observation results of tissue collected 90 days after transplantation in Test Example 9, using CD31 staining and DAPI staining. A: SFT shows tissue collected from the site where adipose tissue obtained by liposuction from a human thigh (living tissue) was transplanted, and C: 3DVFT shows tissue collected from the site where the cell balls from Test Example 9 (1) were transplanted. The upper panel of Figure 13 shows the results observed under bright-field imaging, the middle panel shows the results with CD31 staining, and the lower panel shows the results with DAPI staining. [Modes for carrying out the invention] 【0009】 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. 【0010】 [Cell structure] The cell structure according to this embodiment comprises fragmented extracellular matrix components and cells including at least adipocytes and vascular endothelial cells, and has a vascular network between the cells. 【0011】 Artificially created, thick, three-dimensional tissues are difficult to maintain in the absence of blood vessels and require the supply of oxygen and other nutrients from the outside. In contrast, the cell structure according to this embodiment is expected to be able to be maintained for a long period of time because a network of blood vessels is formed between the cells, similar to that of living tissue. Furthermore, it is expected to be more likely to engraft when transplanted into mammals or other organisms. 【0012】 In this specification, "cellular structure" refers to an aggregate of cells (a cluster of cells) in which cells are arranged three-dimensionally via extracellular matrix components, and is an aggregate artificially created by cell culture. The shape of the cellular structure is not particularly limited, and examples include sheet-like, spherical, nearly spherical, ellipsoidal, nearly ellipsoidal, hemispherical, nearly hemispherical, semicircular, nearly semicircular, rectangular prism-like, nearly rectangular prism-like, etc. Here, biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, etc., and its composition is more complex than that of the cellular structure. Therefore, cellular structures and biological tissue can be easily distinguished. Furthermore, the cellular structure may be aggregated in a cluster while attached to a support, or it may be aggregated in a cluster without being attached to a support. By using multiple cellular structures aggregated in a cluster without being attached to a support, it is possible to efficiently produce cellular tissue in which a vascular network is connected between multiple cellular structures, as will be described later. 【0013】 (cell) 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. An example of adipose-derived stem cell is human adipose-derived stem cell (ADSC). 【0015】 In the cell structure according to this embodiment, the cells include at least adipocytes and vascular endothelial cells. 【0016】 In this specification, "adipocytes" means all adipocytes except adipose stem cells. Adipocytes include mature adipocytes and adipocytes not included in adipose stem cells. Preferably, adipocytes include mature adipocytes, more preferably 90% or more of the total number of adipocytes are mature adipocytes, and even more preferably all are mature adipocytes. Adipocytes may be cells collected from, for example, subcutaneous adipose tissue and epicardial adipose tissue, or collected cells (e.g., adipose stem cells) may be differentiated and used. Adipocytes are not particularly limited, but when adipose tissue constructed from adipocytes is ultimately used to represent tissue in a specific part of the body, it is preferable to use adipocytes derived from tissue corresponding to that part of the body. 【0017】 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. 【0018】 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 cellular structure, and may be 95% or less, 90% or less, 80% or less, or 75% or less. 【0019】 In this specification, "vascular endothelial cells" refers to the flattened cells that make up the surface of the lumen of a blood vessel. Examples of vascular endothelial cells include human umbilical vein endothelial cells (HUVECs). 【0020】 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 cellular structure, and may be 95% or less, 90% or less, 80% or less, or 75% or less. 【0021】 In this embodiment, the cells include at least adipocytes and vascular endothelial cells, but may also include cells other than adipocytes and vascular endothelial cells. Examples of cells other than adipocytes and vascular endothelial 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 (Arota-SMC)), pancreatic islet cells, and keratinocytes (e.g., human epidermal keratinocytes). 【0022】 The ratio of adipocytes to vascular endothelial cells (adipocytes / vascular endothelial cells) in the cell structure according to this embodiment is not particularly limited and may be, for example, 100 / 1 to 1 / 100, 50 / 1 to 1 / 50, 20 / 1 to 1 / 1, 10 / 1 to 1 / 1, 8 / 1 to 1 / 1, 7 / 1 to 1.2 / 1, 6 / 1 to 1.5 / 1, 5 / 1 to 2 / 1, or 3 / 1 to 2 / 1. 【0023】 (Intercellular vascular network) The cell structure according to this embodiment has a vascular network between cells. "Having a vascular network between cells" means having a structure in which branched blood vessels extend between cells so as to surround the cells, similar to living tissue. 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 blood vessel branches and / or the diameter of blood vessels in living tissue. For example, if the average number of blood vessel branches in the cell structure is 80% to 150%, 85% to 130%, or 90% to 120% of the average number of blood vessel branches in living tissue, it may be determined that the number of blood vessel branches is similar to that of living tissue. Alternatively, for example, if the average number of blood vessel branches in the cell structure is 2.5 to 4.5, or 3.0 to 4.2, it may be determined that the number of blood vessel branches is similar to that of living tissue. For example, if the average length between branching vessels in the cell structure 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 the same diversity in diameter as 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 the same diversity in diameter as blood vessels in living tissue. In this embodiment, it is preferable that the cell structure 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 of the cell structure according to this embodiment is 20 μm to 180 μm, or 100 μm to 180 μm, it may be determined that the cell structure has adipocytes similar to those in living tissue.When comparing the above-mentioned biological tissues and cellular structures, the comparison should be 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.). 【0024】 (Fragmented 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 present 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 cell aggregate formation. Specific examples include, but are not limited to, collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, tenascin, entactin, and fibrillin. The extracellular matrix component may be used individually or in combination. For example, the extracellular matrix component may contain collagen components, or may be collagen components. In this embodiment, the extracellular matrix component is preferably a substance present outside animal cells, i.e., an animal extracellular matrix component. The extracellular matrix molecules may be modified or variant forms of the above-mentioned extracellular matrix molecules, or polypeptides such as chemically synthesized peptides, as long as they do not adversely affect cell growth and cell aggregate formation. 【0025】 "Fragmentation" refers to reducing the size of aggregates of extracellular matrix components. Fragmented extracellular matrix components may include defibrated extracellular matrix components. Defibrated extracellular matrix components are those obtained by defibrating the aforementioned extracellular matrix components through the application of physical force. For example, defibration is performed under conditions that do not break the bonds within the extracellular matrix molecules. 【0026】 There are no particular restrictions on the method for fragmenting extracellular matrix components such as collagen components; fragmentation may be carried out by applying physical force. For example, the method for fragmenting extracellular matrix components may involve finely crushing clumps of extracellular matrix components. Extracellular matrix components may be fragmented in a solid phase or in an aqueous medium. For example, extracellular matrix components may be fragmented by applying physical force 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, number of repetitions, etc., it is possible to obtain fragmented extracellular matrix components of millimeter or nanometer size. 【0027】 The diameter and length of fragmented extracellular matrix components can be determined by analyzing the individual fragmented extracellular matrix components using an electron microscope. 【0028】 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, or between 100 μm and 400 μm, from the viewpoint of facilitating the formation of thick tissue. In another embodiment, the average length of the fragmented extracellular matrix components may be 100 μm or less, 50 μm or less, 30 μm or less, 15 μm or less, 10 μm or less, 1 μm or less, or 100 nm or more. It is preferable that the average length of most of the fragmented extracellular matrix components is within the above numerical range. Specifically, it is preferable that the average length of 50% or more of the fragmented extracellular matrix components is within the above numerical range, and it is more preferable that the average length of 95% of the fragmented extracellular matrix components is within the above numerical range. The fragmented extracellular matrix components are preferably fragmented collagen components whose average length is within the above range. 【0029】 The average diameter of the fragmented extracellular matrix components may be 50 nm to 30 μm, 4 μm to 30 μm, or 5 μm to 30 μm. Preferably, the fragmented extracellular matrix components are fragmented collagen components with an average diameter within the above range. 【0030】 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 or the like, and then 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. 【0031】 When the extracellular matrix components are collagen components, the fragmented extracellular matrix components are also called "fragmented collagen components." "Fragmented collagen components" refer to collagen components, such as fibrous collagen components, that have been fragmented while maintaining a triple helix structure. The average length of the fragmented collagen components 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 components is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm. 【0032】 At least some 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. 【0033】 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. 【0034】 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. 【0035】 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. 【0036】 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. 【0037】 When amino groups in extracellular matrix components are used for crosslinking, the degree of crosslinking can be quantified based on the TNBS method described in Non-Patent Document 2, etc. The degree of crosslinking obtained by the TNBS method may be within the above-mentioned range. 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 above-mentioned range. 【0038】 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. 【0039】 The extracellular matrix component content in the cell structure 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. 【0040】 Here, "extracellular matrix components in a cell structure" refers to the extracellular matrix components that constitute the cell structure, and may originate from endogenous extracellular matrix components or from exogenous extracellular matrix components. 【0041】 "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. 【0042】 "Exogenous extracellular matrix components" refers to extracellular matrix components supplied from an external source. The cell structure according to this embodiment includes fragmented extracellular matrix components, which are exogenous extracellular matrix components. The exogenous extracellular matrix components may originate from the same or different animal species as the endogenous extracellular matrix components. Examples of originating animal species include humans, pigs, and cattle. Furthermore, the exogenous extracellular matrix components may be artificial extracellular matrix components. 【0043】 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. 【0044】 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. 【0045】 In other words, when a cell structure contains endogenous extracellular matrix components and fragmented extracellular matrix components, the extracellular matrix component content of the cell structure 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 cell structure and the mass of the decellularized cell structure. 【0046】 For example, if the extracellular matrix component contained in a cell structure is collagen, a method for quantifying the amount of collagen in the cell structure may be the following method for quantifying hydroxyproline. A sample is prepared by mixing hydrochloric acid (HCl) with a lysis solution containing the cell structure, 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 cell structure may be dissolved by directly suspending it in high-concentration hydrochloric acid, centrifuging the lysis solution, and collecting the supernatant, which can then be used for collagen component quantification. Furthermore, the cell structure to be dissolved may be in the state as recovered from the culture medium, or it may be dried after recovery to remove the liquid component before dissolution. However, when quantifying collagen components by dissolving cell structures in their original state after being recovered from the culture medium, the measured weight of the cell structures is expected to vary due to the influence of culture medium components absorbed by the cell structures and residual culture medium due to problems with the experimental procedure. Therefore, from the viewpoint of stably measuring the weight of the tissue and the amount of collagen components per unit weight, it is preferable to use the weight after drying as the basis. 【0047】 More specifically, the following methods can be used to quantify the amount of collagen. (Sample preparation) The entire volume of the freeze-dried cell structure is mixed with 6 mol / L HCl, incubated at 95°C for at least 20 hours in a heat block, and 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 needed so that the results fall within the calibration curve in the measurement described later, 200 μL of the sample is diluted with 100 μL of ultrapure water to prepare the sample. 35 μL of the sample is used. 【0048】 (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. 【0049】 (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. 【0050】 The collagen component within a cell structure may be defined by its area ratio or volume ratio. "Defining by area ratio or volume ratio" means, for example, making the collagen component within the cell structure 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 ​​the collagen component within the entire cell structure 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 cell structure; however, if the cell structure is spherical, for example, it may be defined by a cross-sectional view passing through its approximate center. 【0051】 For example, when defining the collagen component in a cell structure 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 cell structure. The "collagen component in the cell structure" is as described above. The area ratio of the collagen component constituting the cell structure means 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 cell structure 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 cell structure. 【0052】 The cell structure is preferably retained at a rate of 70% or more, more preferably at 80% or more, and even more preferably at 90% or more, after trypsin treatment at a trypsin concentration of 0.25%, temperature of 37°C, pH of 7.4, and reaction time of 15 minutes. Such a cell structure 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 cell structure before and after trypsin treatment. 【0053】 The above cell structure 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 cell structures are less susceptible to enzymatic degradation during or after culture and are stable. 【0054】 The thickness of the above cell structure is preferably 10 μm or more, more preferably 100 μm or more, and even more preferably 1000 μm or more. Such a cell structure has a structure closer to that of 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 cell structure, 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. 【0055】 Here, "thickness of the cell structure" refers to the distance between both ends in a direction perpendicular to the main surface, if the cell structure is sheet-like or rectangular. If the main surface has irregularities, the thickness refers to the distance at the thinnest part of the main surface. 【0056】 Furthermore, if the cell structure is spherical or nearly spherical, it refers to its diameter. Moreover, if the cell structure is ellipsoidal or nearly ellipsoidal, it refers to its minor axis. If the cell 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 cell structure intersects the surface. 【0057】 (Fibrin) The cell structure according to this embodiment may contain fibrin. Fibrin is a component produced when thrombin acts on fibrinogen, releasing A and B chains from the N-terminuses of the Aα and Bβ chains. Fibrin is a polymer and is generally insoluble in water. Fibrin is formed by contacting fibrinogen with thrombin. 【0058】 [Method for manufacturing cell structures] The method for producing a cell structure having a vascular network between cells according to this embodiment includes a contact step of bringing fragmented extracellular matrix components into contact with cells, and a culture step of culturing the cells that have been in contact with the fragmented extracellular matrix. In the contact step, the cells include (i) at least adipocytes, stem cells, and vascular endothelial cells, or (ii) at least adipose stem cells and vascular endothelial cells. 【0059】 (Contact process) In the manufacturing method according to this embodiment, the contact step is a step of bringing fragmented extracellular matrix components into contact with cells. 【0060】 The cells in the contact step are (i) comprising at least adipocytes, stem cells, and vascular endothelial cells, or (ii) comprising at least adipose stem cells and vascular endothelial cells. The cells and each cell are as described above. Cells other than adipocytes, stem cells, and vascular endothelial cells may be included in (i), and cells other than adipose stem cells and vascular endothelial cells may be included in (ii). Stem cells in (i) are preferably adipose stem cells. Furthermore, it is preferable that the adipocytes include mature adipocytes. 【0061】 Fragmented extracellular matrix components, when dispersed in an aqueous medium, can be made to come into contact with cells in the aqueous medium, potentially promoting the formation of cellular structures. 【0062】 In the contact step, extracellular matrix components and cells are brought into contact in an aqueous medium. The contact step may include, 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. 【0063】 Furthermore, there are no particular restrictions on the order in which the cells are brought into contact with the fragmented extracellular matrix components. For example, in the case of (i) above, adipocytes may be added after stem cells and vascular endothelial cells have been added to the aqueous medium containing the fragmented extracellular matrix components, or stem cells, vascular endothelial cells, and adipocytes may be added in that order to the aqueous medium containing the fragmented extracellular matrix components, or stem cells, vascular endothelial cells, and adipocytes may be added simultaneously to the aqueous medium containing the fragmented extracellular matrix components, or the aqueous medium containing the fragmented extracellular matrix components may be added to the aqueous medium containing stem cells, vascular endothelial cells, and adipocytes. After each of the above additions, mixing by stirring or other means is permitted, or mixing is not permitted. The contact step may also include a step of incubating for a certain period of time after the fragmented extracellular matrix components and cells have been brought into contact. 【0064】 The contact step may be performed after the cell layer has been formed in an aqueous medium. In other words, the contact step may be performed by contacting the extracellular matrix component after the cell layer has been formed in an aqueous medium. By forming the cell layer before contacting the extracellular matrix component, a cell structure with a high cell density in the lower layer can be created. 【0065】 Fragmented extracellular matrix components can be obtained by the method described above. Fragmented extracellular matrix components may also be obtained by fragmenting extracellular matrix components in an aqueous medium. That is, the manufacturing method according to this embodiment may include a step of fragmenting extracellular matrix components in an aqueous medium (fragmentation step) before the contact step. The aqueous medium may be the same as the aqueous medium containing the fragmented extracellular matrix components described above. 【0066】 The fragmented extracellular matrix components may be those exemplified above and may include fragmented collagen components. 【0067】 The manufacturing method according to this embodiment may further include a step of heating the extracellular matrix components before the fragmentation step to crosslink at least a portion of the extracellular matrix components, or a step of heating the extracellular matrix components after the fragmentation step and before the contact step to crosslink at least a portion of the extracellular matrix components. 【0068】 In the crosslinking process, the temperature (heating temperature) and time (heating time) for heating the extracellular matrix components can be determined as appropriate. The heating temperature may be, for example, 100°C or higher, 200°C or lower, or 220°C or lower. Specifically, the heating temperature may be, for example, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 220°C, etc. The heating time (the time to hold at the above heating temperature) can be set as appropriate depending on the heating temperature. For example, when heating at 100°C to 200°C, the heating time may be 6 hours or more and 72 hours or less, more preferably 24 hours or more and 48 hours or less. In the crosslinking process, heating may be carried out in the absence of a solvent, or under reduced pressure conditions. 【0069】 The manufacturing method according to this embodiment may include a drying step after the fragmentation step in which the fragmented extracellular matrix components are dried. 【0070】 In the drying process, the defibrated extracellular matrix components are dried. Drying may be carried out, for example, by freeze-drying. By performing the drying process after the defibration process, the aqueous medium is removed from the liquid containing the fragmented extracellular matrix components and the aqueous medium. The removal of the aqueous medium does not mean that there is absolutely no moisture attached to the fragmented extracellular matrix components, but rather that there is no moisture attached to an extent that can be reasonably achieved by the general drying method described above. 【0071】 The stem cell content may be 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 contact process, and may be 95% or less, 90% or less, 80% or less, or 75% or less. 【0072】 The vascular endothelial cell content may be 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 contact process, and may be 95% or less, 90% or less, 80% or less, or 75% or less. 【0073】 The concentration of extracellular matrix components in the contact process can be appropriately determined according to the shape, thickness, and size of the culture vessel of the target cell structure. For example, the concentration of extracellular matrix components in the aqueous medium during the contact process may be 0.1 to 90% by mass or 1 to 30% by mass. 【0074】 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. 【0075】 In the contact process, the mass ratio of extracellular matrix components to cells (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. 【0076】 The ratio of stem cells to vascular endothelial cells (stem cells / vascular endothelial cells) in the contact process is not particularly limited and may be, for example, 100 / 1 to 1 / 100, 50 / 1 to 1 / 50, 20 / 1 to 1 / 1, 10 / 1 to 1 / 1, 8 / 1 to 1 / 1, 7 / 1 to 1.2 / 1, 6 / 1 to 1.5 / 1, 5 / 1 to 2 / 1, or 3 / 1 to 2 / 1. 【0077】 The process may include adding fibrinogen and / or thrombin during the contact step, or after the contact step and before the culture step. When both fibrinogen and thrombin are added, for example, they may be added simultaneously, or fibrinogen may be added first, followed by thrombin. The timing of adding fibrinogen and / or thrombin is not particularly limited; for example, they may be added to an aqueous medium containing stem cells, vascular endothelial cells, adipocytes, and extracellular matrix components, or to an aqueous medium containing stem cells, vascular endothelial cells, and extracellular matrix components. Alternatively, for example, fibrinogen may be added to an aqueous medium containing stem cells, vascular endothelial cells, and extracellular matrix components, followed by adipocytes, and then thrombin. Adding fibrinogen and / or thrombin can suppress shrinkage that may occur in the culture step described later, making it easier to control the shape and size of the cell structure. Furthermore, it can gel the suspension of cells and extracellular matrix components, making it easier to detach the suspension from the culture vessel (support) after dropping it onto the vessel. Furthermore, if the cells include mature adipocytes, the adipocytes may become suspended due to the influence of lipid droplets within the mature adipocytes, potentially resulting in a heterogeneous culture environment between adipocytes and other cells. However, by gelling the suspension, it becomes easier to maintain a uniform mixture of each cell and extracellular matrix component, and to keep the cells and extracellular matrix components in close proximity. 【0078】 The process may further include a step of settling the extracellular matrix components and cells together in an aqueous medium after the contact step and before the culture step. Performing such a step results in a more uniform distribution of extracellular matrix components and cells in the cellular structure. There are no particular limitations on the specific method, but one example is to centrifuge the culture medium containing the extracellular matrix components and cells. 【0079】 (Culture process) In the manufacturing method according to this embodiment, the culture step is a step of culturing cells that have come into contact with fragmented extracellular matrix. 【0080】 There are no particular limitations on the method for culturing cells in contact with fragmented extracellular matrix, and a suitable culture method can be used depending on the type of cells being cultured. For example, the culture 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 culture time may be 1 day to 2 weeks or 1 week to 2 weeks. 【0081】 The culture vessel (support) used for culturing cells in contact with fragmented extracellular matrix 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 the 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. 【0082】 There are no particular restrictions on the culture medium, and a suitable medium can be selected depending on the type of cells being cultured. Examples of suitable media include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The medium may be serum-added or serum-free. The medium may also be a mixed medium, a mixture of two types of media. 【0083】 The cell density in the culture medium during the culture process can be appropriately determined according to the shape, thickness, and size of the culture vessel of the target cell structure. For example, the cell density in the culture medium during the culture process can be 1 to 10 8 It may be cells / mL, 10 3 ~10 7 The cell density may be cells / mL. Furthermore, the cell density in the culture medium during the culture process may be the same as the cell density in the aqueous medium during the contact process. 【0084】 The cell structures produced by the manufacturing method according to this embodiment preferably have a shrinkage rate of 20% or less during culture, more preferably 15% or less, and even more preferably 10% or less. The above shrinkage rate can be calculated, for example, by the following formula. In the formula, L1 represents the length of the longest part of the cell structure on day 1 after culture, and L3 represents the length of the corresponding part of the cell structure on day 3 after culture. Contraction rate (%) = {(L1 - L3) / L1} × 100 【0085】 In the example above, the contraction rate is calculated from the cell structure on day 1 after culturing and the cell tissue on day 3 after culturing. However, it may also be calculated from the cell structure at any point during the culture period, including the end of the culture period. For example, it may be calculated from the cell structure on day 1 after culturing and the cell structure on day 2 after culturing, or from the cell structure on day 1 after culturing and the cell structure on day 5 after culturing, or from the cell structure on day 1 after culturing and the cell structure on day 8 after culturing. 【0086】 After the above culture step (hereinafter also referred to as the "first culture step"; the initial contact step is also referred to as the "first contact step"), the process may include a step of further contacting cells (second contact step) and a step of culturing cells (second culture step). The cells used in the second contact step and the second culture step may be the same species as the cells used in the first contact step and the first culture step, or they may be different species. A two-layered cell structure can be created by the second contact step and the second culture step. Furthermore, by repeatedly including the contact step and the culture step, a multi-layered cell structure can be created, making it possible to create more complex tissues that are closer to living organisms. 【0087】 According to the manufacturing method of this embodiment, a cell structure having a vascular network between cells can be manufactured. The cell structure having a vascular network between cells is as described above. 【0088】 The above culture step may include culturing the cells that have come into contact with the fragmented extracellular matrix in a state where they are not adhered to the support. This makes it possible to produce a cell structure that is aggregated in clumps without being adhered to the support. If the cells that have come into contact with the fragmented extracellular matrix are adhered to the support, the above culture step may include separating the cells that have come into contact with the fragmented extracellular matrix from the support. If the cells that have come into contact with the fragmented extracellular matrix in the above culture step are not adhered to the support from the beginning, culturing them in this state will produce a cell structure that is aggregated in clumps without being adhered to the support. 【0089】 The method for separating cells that have come into contact with the fragmented extracellular matrix from the support is not particularly limited. For example, a low-adhesion support may be used, and cells may be separated from the support by adding culture medium; cells may be physically separated directly from the support using instruments or the like; cells may be separated from the support by applying vibration; or a support coated with a functional material that breaks the bond between the support and cells in response to stimuli such as heat and light may be used, and cells may be separated from the support by applying these stimuli. When separating cells from the support by adding culture medium, the culture medium may be one of the culture media exemplified above. 【0090】 In the culture process, methods for culturing cells that have come into contact with the fragmented extracellular matrix without them being attached to a support from the beginning include, for example, a method in which a suspension containing cells that have come into contact with the fragmented extracellular matrix is ​​gelled and then gently dropped into a culture medium for culturing, and a method in which the shape of cells that have come into contact with the fragmented extracellular matrix is ​​fixed to some extent in a high-viscosity solvent, and then only the solvent is removed and the cells are transferred to a culture vessel. 【0091】 In the culture process described above, the timing for separating the cells in contact with the fragmented extracellular matrix from the support is not particularly limited. For example, it may be done 1 to 7 days after the start of culture, 1 to 24 hours later, 1 to 60 minutes later, 5 to 30 minutes later, or 10 to 20 minutes later. 【0092】 The culture period after separating the cells in contact with the fragmented extracellular matrix from the support is not particularly limited and may be 1 day or more, 1 to 21 days, 3 to 14 days, or 7 to 14 days. 【0093】 [Cell tissue and methods for producing the same] A cell tissue can be produced in which the vascular network is connected between multiple cell structures by using a plurality of cell structures that are aggregated in a clump without being adhered to the support described above, i.e., "cell structures that include fragmented extracellular matrix components and cells, have a vascular network between the cells, and are aggregated in a clump without being adhered to a support, wherein the cells include at least adipocytes and vascular endothelial cells." 【0094】 The production of the above-described cell tissue includes suspension culture of the cell structures that are not adhered to multiple supports. During the suspension culture process, the multiple cell structures adhere to each other, and the vascular network connects between the cell structures, making it easy to produce a large cell tissue with a connected vascular network. The number and size of the cell structures used can be appropriately selected depending on the intended use of the cell tissue, the desired size of the cell tissue, etc. The type of culture medium (culture solution) used for suspension culture and the culture conditions can also be appropriately selected, but for example, the culture medium and conditions exemplified in the above (culture process) can be used. 【0095】 [Uses of cell structures] As described above, the cell structure according to this embodiment has a vascular network formed between cells, similar to living tissue, and is expected to be more likely to engraft when transplanted into animals such as mammals, making it suitable for transplantation. The cell structure used for transplantation may be one or multiple. If multiple are used, for example, 1 to 1000, 10 to 500, or 50 to 200 cells can be used. 【0096】 There are no particular restrictions on the animals that can be transplanted, but they may be mammals, humans, or non-human animals such as monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats. 【0097】 The transplantation method according to this embodiment includes transplanting a cell structure having a vascular structure according to this embodiment into an animal. Prior to transplantation, it may further include preparing the animal to be transplanted and / or preparing the cell structure by the method described above. The transplantation method is not particularly limited and can be carried out by any known surgical method as appropriate according to the target of transplantation, etc. Examples of surgical methods include incising the skin of the target and transplanting directly under the skin, or injecting under the skin of the target using a syringe or the like. The cell structure to be transplanted may be one, multiple, or a cell tissue containing multiple cell structures. The cell structure or cell tissue may be taken from a culture medium, or, depending on the form of transplantation, the cell structure or cell tissue may be gelled or semi-gelled as appropriate (e.g., fibrin gel), or a dispersion in which multiple cell structures are dispersed may be used. Alternatively, a cell structure that has been collected from a culture medium and aggregated into clumps without being attached to multiple supports, with fibrin added, may be used for transplantation. The cell structure according to this embodiment, which includes adipocytes, can be applied, for example, to tissue reconstruction after soft tissue defects caused by trauma, tumor removal, and mastectomy. 【0098】 The vascular network of transplanted cell structures connects to the recipient's own blood vessels around the transplant site. When multiple cell structures are used, which are aggregated in clumps without being attached to a support, the vascular network also connects between the multiple cell structures. Adipose tissue formed in the recipient by transplanting multiple cell structures aggregated in clumps without being attached to a support exhibits superior engraftment because the vascular network connects not only between the multiple cell structures but also to the recipient's own blood vessels. 【0099】 A method for producing a non-human model animal according to this embodiment includes transplanting a cell structure according to this embodiment into a non-human animal. Prior to transplantation, this may further include preparing the non-human animal to be transplanted and / or producing the cell structure by the method described above. The transplantation method is as described above, and the cell structure to be transplanted may be one, multiple, or a cell tissue containing multiple cell structures. After transplanting the cell structure into the non-human animal, the method for producing a non-human model animal according to this embodiment may include growing the animal for, for example, 7 days or more, 30 days or more, or 90 days or more. The non-human model animal according to this embodiment can be used to apply data obtained from animal experiments to humans. To produce a non-human model animal, it is preferable to use a non-human animal in which rejection of the graft has been suppressed, for example, a non-human animal with reduced immunity or immunodeficiency. The non-human model animal can be used, for example, as a pathological in vitro model for inflammatory diseases related to adipose tissue, or for screening of pharmaceuticals for diabetes, obesity, etc., and for assay screening of cosmetics for cellulite, obesity, etc. 【0100】 The cell structure itself according to this embodiment can also be used as a substitute for experimental animals, a transplant material, etc. Specific examples include the tissue reconstruction, pathological in vitro models, drug screening (drug evaluation), and cosmetic assay screening described above. [Examples] 【0101】 <Test Example 1: Preparation of defibrated collagen components> 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). 【0102】 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 (sCMF). The dispersion was freeze-dried by a conventional method to obtain defibrated collagen components (sCMF) as a dried product. The average length of the sCMF was 14.8 ± 8.2 μm (N=20). 【0103】 <Test Example 2: Preparation and Evaluation of Cellular Structures (1)> The cells, reagents, and preparation methods used in the creation of the cell structures are as follows: (Cells and collagen) Human adipose tissue (derived from the thigh) for obtaining primary human mature adipocytes and human adipose stem cells (ADSCs) (provided by Kyoto Prefectural University of Medicine Hospital) • Human umbilical vein-derived vascular endothelial cells (HUVEC) (Lonza Corporation, #C-2517A) • Dialyzed collagen component (sCMF) (produced in Experiment 1) 【0104】 (reagent) • Bovine pancreas-derived insulin (Sigma-America #I1882) • Bovine plasma-derived thrombin freeze-dried powder (Sigma-Ace #T4648) • Collagenase type I derived from Clostridium histolyticum (Sigma-Ace #C0130) • Bovine plasma-derived fibrinogen type IS (Sigma-Ace #F8630) • DMEM (high glucose, Nacalai Tesque) • EGM-2MV Bullet Kit with Growth Factors (Lonza Corporation, #C-2517A) 【0105】 (Culture medium and various solutions) • EGM-2 medium: 500 mL of EBM-2 mixed with EGM-2 supplement growth factors and stored at 4°C. • 10 mg / mL Insulin Stock Solution: Dissolve 100 mg of the above bovine pancreas-derived insulin in 10 mL of 1% glacial acetic acid solution (pH ≤ 2) diluted with water, dispense equal volumes into Eppendorf tubes, and store at -20°C. • 2 mg / mL Collagenase Solution: Mix 2.5 g of BSA with 50 mL of DMEM (0% FBS, 1% antibiotic). To digest all the adipose tissue in a 6-well plate, mix 26 mg of type I collagenase with 13 mL of DMEM (0% FBS, 5% BSA, 1% antibiotic) and filter it through a 0.2 μm pore filter. • 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. • 202U / mL Thrombin Stock Solution: Weigh 202U of thrombin into an Eppendorf tube, immediately add 1mL of DMEM (0% FBS, 1% antibiotic), and dissolve in a 37°C water bath for 3-5 minutes. Then, filter through a 0.2μm pore filter and use by dispensing an equal volume into an Eppendorf tube. 【0106】 (Manufacturing method) Human adipose tissue fragments were washed with PBS containing 5% antibiotic. 4-6 g of tissue was divided into 6 wells of a 6-well plate. Using scissors and tweezers, the tissue was finely chopped into pieces approximately 1-3 mm in size in 2 mL of 2 mg / mL collagenase solution. After incubation at 37°C and 230 rpm for 1 hour, the mixture was mixed with a 10 mL pipette for 30 minutes. The lysate was filtered through a 500 μm pore size iron mesh filter, and all digested cells were collected by adding 2 mL of DMEM per well. The plates were then centrifuged at 200 g for 3 minutes at room temperature (15-25°C). Mature adipocytes were contained in the upper yellow oily layer, while adipose stem cells and blood cells were contained in the pellet. Using a long needle and a 10 mL syringe, the medium between the upper and lower layers was aspirated and discarded. The mature adipocytes in the upper layer and the adipose stem cells and blood cells in the lower layer were washed twice with 25 mL of PBS (5% BSA, 1% antibiotic). During the washing process, the sample was separated into three layers—an upper layer, a lower layer, and a medium between the upper and lower layers—by centrifugation, as described above. The medium between the upper and lower layers was then aspirated and discarded. After two washes, the sample was washed with 25 mL of DMEM. 【0107】 Only the upper layer containing mature adipocytes was collected and dispensed into Eppendorf tubes. The nuclei were stained with Hoechst stain (1000-fold dilution of Hoechst, staining for 15 minutes), and the number of cells was counted using a Turker Burk hemocytometer under a fluorescence microscope. 【0108】 ADSC-containing pellets were suspended in 10 mL of DMEM, seeded in 10 cm dishes, and subcultured. ADSCs were isolated from the dishes using trypsin / EDTA, suspended in 1 mL of DMEM, and the number of cells was counted. 【0109】 HUVEC cells purchased from Lonza were suspended in 10 mL of DMEM, seeded in 10 cm dishes, and subcultured. HUVEC cells were isolated from the dishes using trypsin / EDTA, suspended in 1 mL of DMEM, and the number of cells was counted. 【0110】 1 mg of sCMF was weighed out, 100 μL of DMEM was added, and the mixture was gently mixed until only small particles of sCMF were observed. The mixture was centrifuged at 10,000 rpm for 1 minute at room temperature, and the supernatant was aspirated to obtain an sCMF pellet. 250,000 cells of ADSC and 125,000 cells of HUVEC (ADSC:HUVEC=2:1) ​​were gently added to the sCMF pellet, and without mixing, the mixture was centrifuged at 3,500 rpm for 1 minute at room temperature, and the supernatant was aspirated. 0.3 mg of fibrinogen (6 μL of 50 mg / mL fibrinogen stock solution) was added and gently mixed with the cells and sCMF. 300,000 cells of mature adipocytes were then added and gently mixed. A small amount of DMEM was added as needed to adjust the total volume to 70 μL. Immediately, 0.15 U of thrombin (0.71 μL of 202 U / mL thrombin stock solution) was added and mixed, and then the mixture was slowly seeded into a transwell placed on a 6-well adapter on a 6-well plate. 【0111】 The mixture was incubated in a 37°C incubator for 1 hour to induce gelation, and 12 mL of EGM-2 medium containing insulin at a final concentration of 10 μg / mL was added. The 12 mL of medium was replaced every 2-3 days until day 7 of culture. 【0112】 Fluorescence imaging of the cell structures was performed using the following procedure. The transwells containing the obtained cell structures were transferred to a 24-well plate. After washing with 2 mL of PBS, the tissue was fixed overnight at 4°C using 2 mL of 4% PFA. The cells were washed three times with 2 mL of PBS. The cells were permeabilized with 0.05% Triton / PBS for 7 minutes at room temperature (500 μL in the transwell, 500 μL outside the transwell), and washed three times with 2 mL of PBS. 【0113】 Tissue was blocked for 1 hour at room temperature using a 1% BSA / PBS solution (500 μL in the transwell, 500 μL outside the transwell). The BSA solution was aspirated, and 100 μL of primary antibody solution (CD31 and perilipin diluted 100-fold in 1% BSA / PBS solution) was added (50 μL in the transwell, 50 μL outside the transwell). A wet wipe was placed under the plate, covered with aluminum foil, and incubated overnight at 4°C. After washing three times with 2 mL of PBS, 100 μL of secondary antibody solution (AlexaFluor647-labeled anti-mouse anti-CD31 antibody and AlexaFluor488-labeled anti-rabbit anti-perilipin antibody diluted 200-fold in 1% BSA / PBS solution, and Hoechst diluted 1000-fold) was added (50 μL in the transwell, 50 μL outside the transwell). A wet wipe was placed under the plate, covered with aluminum foil, incubated at room temperature for 2 hours, and then washed four times with 2 mL of PBS. 【0114】 The Transwell membrane was cut, and the gel was placed directly on the bottom of a glass-bottom dish containing a small amount of PBS. The stained cell structures were then observed using a confocal laser scanning microscope (FV3000, Olympus Corporation) with laser excitation light at 640 nm (AlexaFluor647) and 488 nm (AlexaFluor488). 【0115】 The diameter of lipid droplets in the fabricated cell structures was measured using an electron microscope. The diameter of blood vessels and the distance between vascular branches were measured using ImageJ. 【0116】 Figure 1 shows the fluorescence observation results (20x magnification) of a cellular structure with a vascular network. (a) is living tissue, which is adipose tissue taken from a living organism and fixed as is, and (b) is a cellular structure. In the cellular structure, it was confirmed that a vascular network was formed surrounding mature adipocytes (large, round, simple-shaped lipid droplets), similar to living tissue. These vascular networks were formed even into the interior of the cellular structure. The average diameter of the lipid droplets was 85 μm (N=50), which was close to the average diameter of mature adipocytes in living tissue (72 μm (N=50)). Furthermore, the formed blood vessels were hollow, similar to living tissue, and both large diameter vessels (e.g., 10 μm to less than 25 μm) and small diameter vessels (e.g., greater than 0 μm but less than 10 μm) were observed, similar to living tissue. As shown in Figure 2, the number of vascular branches was also found to be close to the number in living tissue. Furthermore, the distribution of the length between blood vessel branches was 0-100 μm: 42.5%, 100-200 μm: 39.4%, 200 μm and above: 18.1% (N=180), which was found to be close to the distribution of the length between blood vessel branches in the vascular network in living tissue (0-100 μm: 61.8%, 100-200 μm: 24.7%, 200 μm and above: 13.5% (N=180)). In Experimental Example 2, the proportion of blood vessel branch lengths between 50 μm and 100 μm was high, and this value is close to the size of mature adipocytes. For example, as explained in J. Silha et al., “Angiogenic factors are elevated in overweight and obese individuals”, International Journal of Obesity (2005) 29, 1308-1314, it is known that in adipose tissue in living organisms, blood vessels surround individual adipocytes, and blood vessels are formed between adipocytes. Given the distribution of the lengths between blood vessel branches as described above, it was suggested that the cell structure in Experiment Example 2 may be faithfully mimicking actual adipose tissue in living organisms. 【0117】 <Test Example 3: Preparation and Evaluation of Cellular Structures (2)> Cell structures were prepared using the same method as in Test Example 2, except that 500,000 mature adipocytes, 2 mg of sCMF, and 0.6 mg of fibrinogen were used. As a result, cell structures with a vascular network surrounding the mature adipocytes were created. Figure 3 shows the fluorescence observation results (10x magnification) of the cell structures stained in the same manner as in Test Example 2. This demonstrates that cell structures with a vascular network can be prepared even when the number of mature adipocytes and the amount of sCMF are changed. 【0118】 <Test Example 4: Preparation and Evaluation of Cellular Structures (3)> Cell structures were created using the same method as in Test Example 2, except that mature adipocytes were not used, and 2 mg of sCMF, 0.6 mg of fibrinogen, and 0.3 U of thrombin were used. As a result, cell structures with a vascular network were created. Figure 4 shows the fluorescence observation results (4x magnification) of the cell structure in which only the blood vessels were stained with anti-CD31 antibody. This demonstrates that cell structures with a vascular network can be created even without using mature adipocytes. 【0119】 <Test Example 5: Preparation and Evaluation of Cellular Structures (4)> Cell structures were created using the same method as in Test Example 2, except that mature adipocytes were not used, and 50,000 HUVEC cells (ADSC:HUVEC=5:1), 2 mg of sCMF, 0.6 mg of fibrinogen, and 0.3 U of thrombin were used. As a result, cell structures with a vascular network were created. Figure 5 shows the fluorescence observation results (4x magnification) of the cell structure in which only the blood vessels were stained with anti-CD31 antibody. This demonstrates that cell structures with a vascular network can be created even without using mature adipocytes and by changing the ratio of ADSC to HUVEC. 【0120】 <Test Example 6: Preparation and Evaluation of Cellular Structures (5)> Cell structures were prepared using the same method as in Test Example 2, except that adipose tissue obtained by liposuction from human thigh (biological tissue) was used instead of mature adipocytes, ADSCs, and HUVECs, and the total volume before seeding was adjusted to 60 μL. The adipose tissue was prepared by finely chopping 3 g of collected biological tissue into pieces of approximately 1-3 mm in size using scissors and forceps, and then liquefying it by slowly pipetting several times using a 10 mL syringe. 60 μL of the liquefied adipose tissue was taken and mixed with sCMF. The adipose tissue obtained by liposuction contains mature adipocytes, adipose stem cells, and vascular endothelial cells. Figure 6 shows the fluorescence observation results (10x magnification) of the cell structures stained using the same method as in Test Example 2. It was shown that cell structures with a vascular network can be prepared even when adipose tissue obtained from biological tissue is used instead of mature adipocytes, ADSCs, and HUVECs. It was also confirmed that cell structures with a vascular network can be prepared similarly when 2 mg of sCMF is used. 【0121】 In all of the experimental examples 2-6, it was confirmed that cell structures with a vascular network could be created. However, comparing experimental examples 2-6, there was a tendency to create cell structures with a vascular network closer to that of living tissue when 1 mg of sCMF was used compared with 2 mg of sCMF. Furthermore, the ratio of ADSC to HUVEC was such that ADSC:HUVEC = 2:1 tended to create cell structures with a vascular network closer to that of living tissue compared with ADSC:HUVEC = 5:1. 【0122】 <Comparative Example> Cell structures were prepared using the same method as in Test Example 2, except that sCMF was not used, and 1 mg of fibrinogen and 0.5 U of thrombin were used. No blood vessel formation was observed in the prepared cell structures. Furthermore, cell structures were prepared using the same method as in Test Example 2, except that ADSC was not included. In the prepared cell structures, only very slight blood vessel formation was observed, and the formation of a vascular network surrounding mature adipocytes could not be confirmed. 【0123】 <Test Example 7: Preparation and Evaluation of Cellular Structures (6)> Similar to Test Example 2, cells, reagents, culture media, and various solutions used in the preparation of cell structures were prepared. The defibrillated collagen component (sCMF) was also prepared using the same method as in Test Example 1. An overview of this test example is shown in Figure 7. In Figure 7, the circles in the droplets on the left represent mature adipocytes, the white diamonds represent ADSCs, and the gray short bars represent HUVECs. 【0124】 (Manufacturing method) Human adipose tissue fragments were washed with PBS containing 5% antibiotic. 4-6 g of tissue was divided into 6 wells of a 6-well plate. Using scissors and tweezers, the tissue was finely chopped into pieces approximately 1-3 mm in size in 2 mL of 2 mg / mL collagenase solution. After incubation at 37°C and 230 rpm for 1 hour, the mixture was mixed with a 10 mL pipette for 30 minutes. The lysate was filtered through a 500 μm pore size iron mesh filter, and all digested cells were collected by adding 2 mL of DMEM per well. The plates were then centrifuged at 200 g for 3 minutes at room temperature (15-25°C). Mature adipocytes were contained in the upper yellow oily layer, while adipose stem cells and blood cells were contained in the pellet. Using a long needle and a 10 mL syringe, the medium between the upper and lower layers was aspirated and discarded. The mature adipocytes in the upper layer and the adipose stem cells and blood cells in the lower layer were washed twice with 25 mL of PBS (5% BSA, 1% antibiotic). During washing, the material was separated into three layers—an upper layer, a lower layer, and a medium between the upper and lower layers—by centrifugation, as described above. The medium between the upper and lower layers was aspirated and discarded. After two washes with 25 mL of PBS (5% BSA, 1% antibiotic), the material was washed with 25 mL of DMEM. 【0125】 Only the upper layer containing mature adipocytes was collected and dispensed into Eppendorf tubes. The nuclei were stained with Hoechst stain (1000-fold diluted Hoechst, stained for 10 minutes), and the number of cells was counted using a Turker Burk hemocytometer under a fluorescence microscope. 【0126】 ADSC-containing pellets were suspended in 10 mL of DMEM, seeded in 10 cm dishes, and subcultured. ADSCs were isolated from the dishes using trypsin / EDTA, suspended in 1 mL of DMEM, and the number of cells was counted. 【0127】 HUVEC cells purchased from Lonza were suspended in 10 mL of DMEM, seeded in 10 cm dishes, and subcultured. HUVEC cells were isolated from the dishes using trypsin / EDTA, suspended in 1 mL of DMEM, and the number of cells was counted. 【0128】 To obtain one roughly spherical cell structure (hereinafter also referred to as a "cell ball") with a diameter of approximately 1 mm, 16,250 mature adipocytes, 13,750 ADSCs, 6,875 HUVECs, 0.06 mg of sCMF, 0.04 mg of fibrinogen, and 0.02 U of thrombin were used. 【0129】 2.4 mg of sCMF was weighed out, 1 mL of DMEM was added, and the mixture was gently mixed until only small particles of sCMF were observed. The mixture was centrifuged at 10,000 rpm for 1 minute at room temperature, and the supernatant was aspirated to obtain an sCMF pellet. 220,000 ADSC cells and 275,000 HUVEC cells were gently added to the sCMF pellet, and the mixture was centrifuged at 3,500 rpm for 1 minute at room temperature, and the supernatant was aspirated. Fibrinogen (50 mg / mL fibrinogen stock solution, 32 μL) was added and gently mixed with the cells and sCMF. 650,000 mature adipocyte cells were then added and gently mixed. A small amount of DMEM was added to adjust the total volume to 200 μL. Immediately, thrombin (202 U / mL thrombin stock solution, 3.9 μL) was added, and after a little mixing, the mixture (enough for 40 cell balls) was seeded into each well of a low-adhesion 96-well plate (IWAKI #4860-800LP) at a rate of 5 μL (enough for 1 cell ball). 【0130】 The mixture was incubated in a 37°C incubator for 15 minutes to induce gelation, and 300 μL of EGM-2 medium containing insulin at a final concentration of 10 μg / mL was added. 【0131】 After 24 hours of incubation, the cells were transferred to a low-adhesion 24-well plate (IWAKI #4820-800LP) and detached from the plate by adding 2 mL of the above EGM-2 medium. The medium was changed every 2-3 days until day 7 of incubation. 【0132】 Fluorescence imaging of cell structures was performed using the following procedure. The transwells containing the obtained cell structures were transferred to a 24-well plate (IWAKI #4820-800LP). After washing with 200 μL of PBS, the tissue was fixed overnight at 4°C using 200 μL of 4% PFA. Three washes were performed using 200 μL of PBS. For immunostaining, the cells were permeabilized with 200 μL of 0.05% Triton / PBS for 7 minutes at room temperature, and then washed three times with 200 μL of PBS. 【0133】 Tissue was blocked for 1 hour at room temperature using 200 μL of 1% BSA / PBS solution. The BSA solution was aspirated, and 100 μL of primary antibody solution (CD31 and perilipin diluted 100-fold in 1% BSA / PBS solution) was added. A wet wipe was placed under the plate, covered with aluminum foil, and incubated overnight at 4°C. After washing three times with 200 μL of PBS, secondary antibody solution (AlexaFluor647-labeled anti-mouse anti-CD31 antibody and AlexaFluor488-labeled anti-rabbit anti-perilipin antibody diluted 200-fold in 1% BSA / PBS solution, and Hoechst diluted 1000-fold) was added. A wet wipe was placed under the plate, covered with aluminum foil, and incubated for 2 hours at room temperature, then washed four times with 200 μL of PBS. 【0134】 Cell balls were placed directly onto a complete plate of a confocal quantitative image cytometer CQ1 (manufactured by Yokogawa Electric), and the stained cell structures were observed using the CQ1 confocal quantitative image cytometer with laser excitation light at 640 nm (AlexaFluor647) and 488 nm (AlexaFluor488). 【0135】 Figure 8 shows the fluorescence observation results of cell balls with vascular networks using Nile Red staining and CD31 staining. CD31 staining was performed in the same manner as in Experiment 2, and Nile Red staining was performed using the standard method. The photograph on the right is a further magnified view of one of the cell balls on the left. It was confirmed that a vascular network was formed in the cell ball, surrounding mature adipocytes (large, round, simple-lenticular lipid droplets), similar to biological tissue. These vascular networks were formed even into the interior of the cell ball. 【0136】 Figure 9 shows the results of immunohistochemical staining of a cell ball containing a vascular network, as observed in the cell ball. This result also confirms that many lumens are observed within the cell ball, indicating that the vascular network extends deep into the cellular structure. 【0137】 Figure 10 shows the average diameter (n=12 cell balls / volume) of cell balls prepared using the method described in this test example after 7 days of culture. The average diameter of 5 μL cell balls was 1256 μm, and the average diameter of 10 μL cell balls was 1857 μm. 【0138】 <Test Example 8: Preparation and Evaluation of Cell Tissue Containing Multiple Cell Balls> Multiple 5 μL cell balls prepared in Test Example 7, cultured for 7 days, were placed adjacent to each other in contact and cultured in 10 mL of culture medium for 7 days in suspension. They were stained and observed in the same manner as in Test Example 7. As a result, as shown in Figure 11, a cellular tissue was obtained in which multiple cell balls (5 in Figure 11) had aggregated and merged. It was confirmed that in the cellular tissue formed by the fusion of the 5 cell balls, not only were vascular networks connected within each cell ball, but vascular networks were also connected between the multiple cell balls (Figure 11 left and upper right). 【0139】 <Test Example 9: Cell Ball Transplantation Test and Evaluation> Six immunodeficient mice were prepared, their back skin was incised, and (1) to (3) below were injected into the incisions, respectively. After suturing, three mice were raised for 30 days and three mice for 90 days. (1) A mixture obtained by suspending 111 cell balls after culture, prepared in Test Example 7, in 100 μL of a solution containing 2.5 mg fibrinogen and 1.25 U thrombin. (2) A mixture prepared in the same manner as in (1), except that HUVEC is not used, by suspending 111 cell balls in 100 μL of a solution containing 2.5 mg fibrinogen and 1.25 U thrombin. (3) Cell structure (100 μL) prepared using adipose tissue obtained by liposuction from a human thigh (biological tissue) in Test Example 6. 【0140】 Tissue samples were collected from the transplant sites of each individual after 30 days of growth. Adipose tissue was formed at the sites where (2) was transplanted, but no vascular network was observed. Adipose tissue was formed at the sites where (3) was transplanted, and a slight vascular network was observed on the tissue surface, but the presence of large oil droplets was also observed. The formation of oil droplets indicates the death of adipocytes. On the other hand, at the sites where (1) was transplanted using the cell balls prepared in Experimental Example 7, adipose tissue with a vascular network covering the tissue surface was observed. Furthermore, no oil droplet formation was observed in the tissue at the sites where (1) was transplanted, indicating superior post-transplant engraftment. 【0141】 Tissue samples were taken from the transplant sites described in (1) above after 30 days of growth and stained with perilipin and DAPI in the same manner as in Test Example 2 (Figure 12). DAPI staining was performed using a standard method. Note that A:SFT shows tissue taken from the transplant sites described in (3) above, and C:3DVFT shows tissue taken from the transplant sites described in (1) above. The upper panel of Figure 12 shows the results observed under bright-field imaging, the middle panel shows the results of perilipin staining, and the lower panel shows the results of DAPI staining. Mature adipocytes were confirmed to have formed in the tissue formed at the transplant sites. 【0142】 Furthermore, tissue was collected from the transplant site described in (1) above from individuals after 90 days of growth and stained with perilipin and CD31 in the same manner as in Test Example 2 (Figure 13). The upper panel of Figure 13 shows the results observed under bright-field conditions, the middle panel shows the results of CD31 staining, and the lower panel shows the results of DAPI staining. It was confirmed that not only were mature adipocytes formed in the tissue formed at the transplant site, but a vascular network was also formed. 【0143】 Based on the above, it has been demonstrated that the cell structure according to this embodiment exhibits excellent engraftment after transplantation and is suitable for transplantation.

Claims

[Claim 1] It comprises fragmented extracellular matrix components and cells, A cellular structure having a vascular network between cells, The cells include at least adipocytes and vascular endothelial cells, The fragmented extracellular matrix component includes collagen, The average length of the fragmented extracellular matrix components is between 100 nm and 400 μm. A gelled cell structure for transplantation. [Claim 2] The transplantable cell structure according to claim 1, wherein the vascular network is formed between the adipocytes. [Claim 3] The transplantable cell structure according to claim 1 or 2, wherein the adipocytes include mature adipocytes. [Claim 4] A cell structure for transplantation according to any one of claims 1 to 3, wherein the extracellular matrix component content in the cell structure is 0.01 to 90% by mass, based on the dry weight of the cell structure. [Claim 5] A cell structure for transplantation according to any one of claims 1 to 4, further comprising fibrin. [Claim 6] A contact step of bringing fragmented extracellular matrix components into contact with cells, wherein the cells (i) include at least adipocytes, stem cells, and vascular endothelial cells, or (ii) include at least adipocytes and vascular endothelial cells, A culture process in which cells in contact with fragmented extracellular matrix are cultured. Includes, The fragmented extracellular matrix component includes collagen, The average length of the fragmented extracellular matrix components is between 100 nm and 400 μm. A method for producing a gelled cell structure having a vascular network between cells. [Claim 7] The method according to claim 6, wherein the cells include adipocytes, adipose stem cells, and vascular endothelial cells. [Claim 8] The method according to claim 6 or 7, wherein the adipocytes include mature adipocytes. [Claim 9] The amount of the fragmented extracellular matrix component in the contact step is 1.0 × 10 ６ The method according to any one of claims 6 to 8, wherein the amount is 0.1 to 100 mg relative to the cells of cells. [Claim 10] The method according to any one of claims 6 to 9, wherein the ratio of stem cells to vascular endothelial cells in the contact step is 100 / 1 to 1 / 100. [Claim 11] The method according to any one of claims 6 to 9, further comprising adding fibrinogen during the contact step, or after the contact step and before the culture step.

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