Cell construct and cell construct production method

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 producing a cell structure, in particular to a cell structure having a vascular network between cells and a method for producing a cell structure having a vascular network between cells. [Background technology]

[0002] As a method for artificially producing a structure that mimics a living tissue, for example, a method for producing a three-dimensional tissue (Patent Document 1) including arranging cells coated with a membrane containing collagen in three dimensions to form a three-dimensional tissue, and a method for producing a three-dimensional cell tissue (Patent Document 2) 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 are known. In addition, the present inventors have proposed a method for producing a large-sized three-dimensional tissue with a thickness of 1 mm or more with a relatively small number of cells by contacting cells with fragmented exogenous collagen (Patent Document 3). Such three-dimensional tissues are expected to be used as substitutes for laboratory animals, transplant 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 [Problem to be solved by the invention]

[0004] According to the above-mentioned method for producing a three-dimensional tissue, a thick three-dimensional tissue can be produced. However, a method for producing adipose tissue in which a vascular network is formed between cells like in biological tissue has not been known.

[0005] Therefore, an object of the present invention is 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] That is, the present invention relates to, for example, the following inventions. [1] A method for producing a tissue-derived tissue comprising the steps of: (1) comprising: A cell structure having a vascular network between cells, The cell structure, wherein the cells include at least adipocytes and vascular endothelial cells. [2] The cell structure according to [1], wherein the vascular network is formed between the fat cells. [3] The cell structure according to [1] or [2], wherein the adipocytes include mature adipocytes. [4] The cell structure according to any one of [1] to [3], wherein the average length of the fragmented extracellular matrix component is 100 nm or more and 400 μm or less. [5] The cell structure according to any one of [1] to [4], wherein the content of extracellular matrix components in the cell structure is 0.01 to 90 mass % based on the dry weight of the cell structure. [6] The cell structure according to any one of [1] to [5], wherein the fragmented extracellular matrix component comprises collagen. [7] The cell structure according to any one of [1] to [6], further comprising fibrin. [8] The cell structure according to any one of [1] to [6], which is for transplantation. [9] A step of contacting a fragmented extracellular matrix component with a cell, the cell comprising (i) at least an adipocyte, a stem cell, and a vascular endothelial cell, or (ii) at least an adipocyte, a stem cell, and a vascular endothelial cell; A culture step of culturing cells in contact with the fragmented extracellular matrix; A method for producing a cell structure having a vascular network between cells, comprising:

[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 components in the contacting step is 1.0 × 10 6 The method according to any one of [9] to

[11] , wherein the amount of the administered dose is 0.1 to 100 mg per 100 cells.

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

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

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

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

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

[14] , further comprising adding fibrinogen after the contacting step, or after the contacting step and before the culturing step.

[16] A non-human model animal having the cell structure according to any one of [1] to [8] as a transplant.

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

[19] A method for producing a tissue-derived tissue comprising fragmented extracellular matrix components and cells, It has a vascular network between the cells. A cell structure that is aggregated in a mass form without being attached to a support, The cell structure, wherein the cells include at least adipocytes and vascular endothelial cells.

[20] The cell structure according to

[19] , which is approximately spherical.

[21] A step of contacting a fragmented extracellular matrix component with a cell, the cell comprising (i) at least an adipocyte, a stem cell, and a vascular endothelial cell, or (ii) at least an adipocyte stem cell and a vascular endothelial cell; A culture step of culturing cells in contact with the fragmented extracellular matrix; Including, A method for producing a cell structure having a vascular network between cells, wherein the culture step includes culturing cells in contact with the fragmented extracellular matrix in a non-adherent state to a support.

[22] The method according to

[21] , wherein the culturing step includes detaching the cells in contact with the fragmented extracellular matrix from the support.

[23] A cellular 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] , which comprises transplanting the cell structure into a non-human animal and then growing the cell structure for 30 days or more.

[27] The method according to

[26] , which comprises transplanting the cell structure into a non-human animal and then growing the cell structure for 90 days or more. Effect 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] 1 shows photographs depicting the results of observation of (a) biological tissue and (b) the cell structure of Test Example 2 by perilipin staining and CD31 staining. [Diagram 2] 1 is a graph comparing the number of blood vessel branches in the cell structure of Test Example 2 with the number of blood vessel branches in biological tissue. [Diagram 3] 1 shows photographs depicting the observation results of the cell structure of Test Example 3 by perilipin staining and CD31 staining. [Figure 4] 1 shows photographs depicting the observation results of the cell structure of Test Example 4 by CD31 staining. [Diagram 5] 1 is a photograph showing the observation results of the cell structure of Test Example 5 by CD31 staining. [Figure 6] 1 shows photographs depicting the observation results of the cell structure of Test Example 6 by perilipin staining and CD31 staining. [Figure 7] Fig. 1 shows an overview of Test Example 7. In the droplet on the left, the circles represent mature adipocytes, the white diamonds represent ADSCs, and the short grey bars represent HUVECs. [Figure 8] Photographs showing the results of fluorescent observation of a cell ball having a vascular network in Test Example 7, using Nile Red staining and CD31 staining. The photograph on the right is a further enlargement of one of the cell balls on the left. [Figure 9] 13 is a photograph showing the observation results of a cell ball having a vascular network by CD31 staining. [Figure 10] 1 is a graph showing the average diameter (n=12 cell balls / amount) of cell balls produced by the method of Test Example 7 after culturing for 7 days. [Figure 11] Photographs showing the results of fluorescent observation of cell tissue prepared by the method of Test Example 8 using Nile Red staining and CD31 staining (left, upper right are partially enlarged photographs) and a photograph of aggregated cell balls on a plate observed in bright field (bottom right). [Figure 12] 12 is a photograph showing the results of fluorescent observation, by perilipin staining and DAPI staining, of tissue taken 30 days after transplantation in Test Example 9. A: SFT shows tissue taken from the site where adipose tissue obtained by liposuction from a human thigh (living tissue) was transplanted, and C: 3DVFT shows tissue taken from the site where the cell ball of Test Example 9 (1) was transplanted. The top row of Fig. 12 shows the results of bright field observation, the center row shows the results of perilipin staining, and the bottom row shows the results of DAPI staining. [Figure 13]13 is a photograph showing the results of fluorescent observation of tissues by CD31 staining and DAPI staining, taken 90 days after transplantation in Test Example 9. A: SFT shows tissue taken from the site where fat tissue obtained by liposuction from a human thigh (living tissue) was transplanted, and C: 3DVFT shows tissue taken from the site where the cell ball of Test Example 9 (1) was transplanted. The top row of Fig. 13 shows the results of bright field observation, the center row shows the results of CD31 staining, and the bottom row shows the results of DAPI staining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0010] [Cell structure] The cell structure according to this embodiment contains fragmented extracellular matrix components and cells including at least adipocytes and vascular endothelial cells, and has a vascular network among the cells.

[0011] It is difficult to maintain an artificially created three-dimensional tissue structure with a certain thickness in the absence of blood vessels, and it is considered necessary to supply oxygen and the like from the outside. In contrast, the cell structure according to the present embodiment is expected to be able to be maintained for a long period of time because a vascular network is formed between the cells like in living tissue. It is also expected that the cell structure will easily take root when transplanted into a mammal or the like.

[0012] In the present specification, the term "cell structure" refers to an assembly of cells (agglomerated cell population) in which cells are arranged three-dimensionally via extracellular matrix components, and is an assembly artificially produced by cell culture. The shape of the cell structure is not particularly limited, and examples thereof include sheet-like, spherical, approximately spherical, ellipsoidal, approximately ellipsoidal, hemispherical, approximately hemispherical, semicircular, approximately semicircular, rectangular, and approximately rectangular. Here, biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, and the like, and has a more complicated structure than cell structures. Therefore, cell structures and biological tissues can be easily distinguished. In addition, the cell structure may be an aggregate in a state of being adhered to a support, or may be an aggregate in a state of being not adhered to a support. By using a plurality of cell structures aggregated in a state of being not adhered to a support, a cell tissue in which a vascular network is connected between a plurality of cell structures can be efficiently produced, as described below.

[0013] (cell) As used herein, the term "cell" is not particularly limited, and may be, for example, a cell derived from a mammal such as a human, monkey, dog, cat, rabbit, pig, cow, mouse, or rat. The site of origin of the cell is also not particularly limited, and may be a somatic cell derived from bone, muscle, internal organs, nerve, brain, bone, skin, blood, or the like, or may be a germ cell. Furthermore, the cell may be a stem cell, or may be a cultured cell such as a primary cultured cell, a subcultured cell, or a cell line cell.

[0014] As used herein, the term "stem cell" refers to a cell having self-renewal and pluripotency. Stem cells include pluripotent stem cells capable of differentiating into any cell type, and tissue stem cells (also called somatic stem cells) capable of differentiating into a specific cell type. 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 stem cells, bone marrow-derived stem cells), hematopoietic stem cells, and neural stem cells. Examples of adipose stem cells include human adipose stem cells (ADSCs).

[0015] In the cell structure according to this embodiment, the cells include at least adipocytes and vascular endothelial cells.

[0016] In the present specification, "adipocytes" refers to all adipocytes except adipose stem cells. Adipocytes include mature adipocytes and adipocytes not included in adipose stem cells, and preferably 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 epicardium-derived adipose tissue, or collected cells (e.g., adipose stem cells) may be induced to differentiate and used. Adipocytes are not particularly limited, but when the adipose tissue constructed from adipocytes is ultimately used as a tissue of a specific part of the body, it is preferable to use those derived from the tissue corresponding to the tissue of that part.

[0017] The size of lipid droplets can be used as an index of the maturity of fat cells. Lipid droplets are intracellular organelles that store lipids such as triglycerides (neutral fats) and cholesterol, and have a droplet-like shape because the lipids are covered with a single membrane of phospholipids. In addition, expression of proteins (perilipin, etc.) specific to adipose tissue is observed on the surface of the phospholipids. The size of lipid droplets in mature fat cells varies, but for example, when the average value of the lipid droplet size is 20 μm or more, it can be said that the fat cells are somewhat mature, that is, mature fat cells.

[0018] The fat 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 of the total number of cells in the cell structure, and may be 95% or less, 90% or less, 80% or less, or 75% or less.

[0019] As used herein, the term "vascular endothelial cells" refers to flat cells that constitute the surface of the blood vessel lumen. Examples of vascular endothelial cells include human umbilical vein-derived vascular endothelial cells (HUVEC).

[0020] The content of vascular endothelial cells may be, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more of the total number of cells in the cell 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 fat cells and vascular endothelial cells, but may include cells other than fat cells and vascular endothelial cells. Examples of cells other than fat cells and vascular endothelial cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, cancer cells such as colon cancer cells (e.g., human colon cancer cells (HT29)), and hepatic cancer cells, cardiac muscle cells, epithelial cells (e.g., human gingival epithelial cells), lymphatic endothelial cells, nerve cells, dendritic cells, hepatic cells, adhesive 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 the number of adipocytes to vascular endothelial cells (adipocytes / vascular endothelial cells) in the cell structure of 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] (vascular network between cells) The cell structure according to the present embodiment has a vascular network between cells. "Having a vascular network between cells" means that, like living tissue, the structure has a structure in which branched blood vessels extend between cells so as to surround the cells. Whether or not a vascular network similar to that of living tissue is formed can be determined based on, for example, the number of blood vessel branches in the living tissue and / or the length between the blood vessel branches and / or the diversity of blood vessel diameters. For example, when the average value of the number of blood vessel branches in the cell structure is 80% or more and 150% or less, 85% or more and 130% or less, or 90% or more and 120% or less, it may be determined that the number of blood vessel branches in the cell structure is similar to that in living tissue. In addition, for example, when the average value of the number of blood vessel branches in the cell structure is 2.5 or more and 4.5 or less, or 3.0 or more and 4.2 or less, it may be determined that the number of blood vessel branches in the cell structure is similar to that in living tissue. For example, when the average value of the length between the branches of blood vessels in the cell structure is 80% or more and 150% or less, 85% or more and 130% or less, or 90% or more and 120% or less, it may be determined that the length between the branches of blood vessels in the cell structure is similar to that in the biological tissue. In the biological tissue, both thick and thin blood vessels are observed. Therefore, for example, when both blood vessels with a thick diameter (e.g., 10 μm or more and less than 25 μm) and blood vessels with a thin diameter (e.g., more than 0 μm and less than 10 μm) are observed as in the biological tissue, it may be determined that the blood vessels have a similar diversity to the diameter of blood vessels in the biological tissue. In addition, when 60% or more, 70% or more, or 80% or more of the total blood vessel diameter is distributed in the range of more than 0 μm and less than 25 μm, it may be determined that the blood vessels have a similar diversity to the diameter of blood vessels in the biological tissue. It is preferable that the cell structure according to this embodiment has a vascular network between fat cells. In that case, it is preferable that not only the blood vessel network but also the fat cells surrounded by the blood vessels are similar to those in the biological tissue. For example, when the average size of the fat droplets of the fat cells in the cell structure of this embodiment is 20 μm to 180 μm, or 100 μm to 180 μm, it may be determined that the cell structure has fat cells similar to fat cells in biological tissue.When comparing the biological tissue and the cell structure, the biological tissue and the cell structure are compared under the same conditions (for example, per fixed volume, per fixed area in the case of image analysis, per fixed sample, etc.).

[0024] (Fragmented extracellular matrix components) In the present specification, the term "extracellular matrix component" refers to an assembly of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. The extracellular matrix refers to a substance that exists outside cells 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 alone or in combination. The extracellular matrix component may, for example, contain a collagen component or may be a collagen component. The extracellular matrix component in this embodiment is preferably a substance that exists outside animal cells, that is, an animal extracellular matrix component. In addition, the extracellular matrix molecule may be a modified or variant of the above-mentioned extracellular matrix molecule, or may be a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect cell growth and cell aggregate formation.

[0025] "Fragmentation" refers to breaking down an aggregate of an extracellular matrix component into smaller sizes. The fragmented extracellular matrix component may include a defibrated extracellular matrix component. The defibrated extracellular matrix component is a component obtained by defibrating the above-mentioned extracellular matrix component by application of a physical force. For example, defibration is performed under conditions that do not break the bonds within the extracellular matrix molecules.

[0026] The method for fragmenting the extracellular matrix components such as collagen components is not particularly limited, and may be fragmented by applying a physical force. The method for fragmenting the extracellular matrix components may be, for example, a method for finely breaking down the clumped extracellular matrix components. The extracellular matrix components may be fragmented in a solid phase or in an aqueous medium. For example, the extracellular matrix components may be fragmented by applying a physical force using an ultrasonic homogenizer, an agitation homogenizer, a high-pressure homogenizer, or the like. When using an agitation homogenizer, the extracellular matrix components may be homogenized as they are, or may be homogenized in an aqueous medium such as physiological saline. It is also possible to obtain fragmented extracellular matrix components of millimeter size or nanometer size by adjusting the homogenization time, number of times, etc.

[0027] The diameter and length of the fragmented extracellular matrix components can be determined by analyzing the individual fragmented extracellular matrix components by electron microscopy.

[0028] The average length of the fragmented extracellular matrix components may be 100 nm or more and 400 μm or less, or 100 nm or more and 200 μm or less. In one embodiment, the average length of the fragmented extracellular matrix components may be 5 μm or more and 400 μm or less, 10 μm or more and 400 μm or less, or 100 μm or more and 400 μm or less, 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 the majority of the fragmented extracellular matrix components among all 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-mentioned range, and it is more preferable that the average length of 95% of the fragmented extracellular matrix components is within the above-mentioned range. The fragmented extracellular matrix components are preferably fragmented collagen components having an average length within the above-mentioned range.

[0029] The average diameter of the fragmented extracellular matrix component may be 50 nm to 30 μm, 4 μm to 30 μm, or 5 μm to 30 μm. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average diameter within the above range.

[0030] The average length and average diameter of the fragmented extracellular matrix components can be determined by measuring each fragmented extracellular matrix component using an optical microscope or the like and analyzing the images. In this specification, "average length" means the average value of the length in the longitudinal direction of the measured sample, and "average diameter" means the average value of the length in the direction perpendicular to the longitudinal direction of the measured sample.

[0031] When the extracellular matrix component is a collagen component, the fragmented extracellular matrix component is also called a "fragmented collagen component." The "fragmented collagen component" refers to a collagen component, such as a fibrous collagen component, that is fragmented and maintains a triple helix structure. The average length of the fragmented collagen component is preferably 100 nm to 200 μm, more preferably 22 μm to 200 μm, and even more preferably 100 μm to 200 μm. The average diameter of the fragmented collagen component is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0032] At least a portion of the fragmented extracellular matrix components may be intermolecularly or intramolecularly crosslinked. The extracellular matrix components may be crosslinked intramolecularly or intermolecularly among the extracellular matrix molecules that make up the extracellular matrix components.

[0033] Examples of the crosslinking method include physical crosslinking by applying heat, ultraviolet light, radiation, etc., and chemical crosslinking by a crosslinking agent, enzyme reaction, etc., but the method is not particularly limited. From the viewpoint of not interfering with cell growth, physical crosslinking is preferred. Crosslinking (physical crosslinking and chemical crosslinking) may be crosslinking via a covalent bond.

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

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

[0036] The degree of crosslinking can be determined appropriately depending on the type of extracellular matrix component, the means of crosslinking, 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 be 30% or less, 20% or less, or 15% or less. When the degree of crosslinking is 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 the 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 and the like. The degree of crosslinking measured by the TNBS method may be within the above-mentioned range. The degree of crosslinking measured by the TNBS method is the proportion of amino groups used for crosslinking among the amino groups in the extracellular matrix. When the extracellular matrix components contain 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 a water-insoluble extracellular matrix component, the degree of crosslinking may be quantified by the TBO (toluidine blue O) method. The degree of crosslinking by the TBO method may be within the above-mentioned range.

[0039] The content of extracellular matrix components in the cell structure may be 0.01 to 90 mass% based on the above cell structure (dry weight), preferably 10 to 90 mass%, preferably 10 to 80 mass%, preferably 10 to 70 mass%, preferably 10 to 60 mass%, preferably 1 to 50 mass%, preferably 10 to 50 mass%, more preferably 10 to 30 mass%, and more preferably 20 to 30 mass%.

[0040] Here, "extracellular matrix components in a cell structure" means extracellular matrix components that constitute the cell structure, and may be derived from endogenous extracellular matrix components or 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 the above-mentioned mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts. Endogenous extracellular matrix components may be fibrous or non-fibrous.

[0042] "Exogenous extracellular matrix components" refers to extracellular matrix components supplied from the outside. The cell structure according to this embodiment contains 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 the animal species from which the exogenous extracellular matrix components originate include humans, pigs, and cows. The exogenous extracellular matrix components may also be artificial extracellular matrix components.

[0043] When the extracellular matrix component is a collagen component, the exogenous extracellular matrix component is also called an "exogenous collagen component", and the "exogenous collagen component" means a collagen component supplied from the outside, which is an aggregate of collagen molecules formed by a plurality of collagen molecules, and specifically includes fibrous collagen, non-fibrous collagen, etc. The exogenous collagen component is preferably fibrous collagen. The fibrous collagen means a collagen component that is the main component of collagen fibers, and examples thereof include type I collagen, type II collagen, and type III collagen. The fibrous collagen may be a commercially available collagen, and a specific example thereof is type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. An example of an exogenous non-fibrous collagen is type IV collagen.

[0044] The exogenous extracellular matrix component may be derived from an animal species different from that of the cells. Also, when the cells include extracellular matrix-producing cells, the exogenous extracellular matrix component may be derived from an animal species different from that of the extracellular matrix-producing cells. That is, the exogenous extracellular matrix component may be a xenogeneic extracellular matrix component.

[0045] That is, when a cell structure contains endogenous extracellular matrix components and fragmented extracellular matrix components, the content of extracellular matrix components constituting the cell structure means the total amount of the endogenous extracellular matrix components and the fragmented extracellular matrix components. The content of the extracellular matrix can be calculated from the volume of the obtained cell structure and the mass of the decellularized cell structure.

[0046] For example, when the extracellular matrix component contained in the cell structure is a collagen component, the method for quantifying the amount of collagen component in the cell structure includes, for example, the following method for quantifying hydroxyproline. A lysis solution in which the cell structure is dissolved is mixed with hydrochloric acid (HCl), incubated at high temperature for a predetermined time, returned to room temperature, centrifuged, and the supernatant is diluted to a predetermined concentration to prepare a sample. A hydroxyproline standard solution is treated in the same manner as the sample, and then diluted stepwise to prepare a standard. Each of the sample and the standard is treated as specified with a hydroxyproline assay buffer and a detection reagent, and the absorbance at 570 nm is measured. The amount of collagen component is calculated by comparing the absorbance of the sample with the standard. The cell structure may be directly suspended in high-concentration hydrochloric acid, the lysis solution is centrifuged, and the supernatant is collected and used for collagen component quantification. The cell structure to be dissolved may be in the state as it is recovered from the culture solution, or may be dissolved in a state in which the liquid components are removed by drying after recovery. However, when quantifying collagen components by dissolving cell structures in the state recovered from the culture medium, it is expected that the measured weight of the cell structure will vary due to the influence of medium components absorbed by the cell structure and residual medium due to problems with the experimental technique. 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 method for quantifying the amount of collagen component may be, for example, the following method. (Sample preparation) The entire amount of the freeze-dried cell structure is mixed with 6 mol / L HCl, incubated at 95°C in a heat block for more than 20 hours, and then returned to room temperature. After centrifugation at 13,000g for 10 minutes, the supernatant of the sample solution is collected. After diluting appropriately with 6 mol / L HCl so that the results fall within the range of the calibration curve in the measurement described below, the sample is prepared by diluting 200 μL with 100 μL of ultrapure water. 35 μL of sample is used.

[0048] (Preparation of standards) Add 125μL of standard solution (1200μg / mL in acetic acid) and 125μL of 12mol / l HCl to a screw-cap tube, mix, incubate in a heat block at 95℃ for 20 hours, then return to room temperature. After centrifugation at 13000g for 10 minutes, dilute the supernatant with ultrapure water to make S1 at 300μg / mL, and gradually dilute S1 to make S2 ​​(200μg / mL), S3 (100μg / mL), S4 (50μg / mL), S5 (25μg / mL), S6 (12.5μg / mL), and S7 (6.25μg / mL). Also prepare S8 (0μg / mL) containing only 90μL of 4mol / l HCl.

[0049] Assay Add 35 μL of each standard and sample to the plate (included in the QuickZyme Total Collagen Assay kit, QuickZyme Biosciences). Add 75 μL of assay buffer (included in the above kit) to each well. Seal the plate and incubate at room temperature for 20 minutes with shaking. Remove the seal and add 75 μL of detection reagent (reagent A:B=30 μL:45 μL, included in the above kit) to each well. Seal the plate, mix the solution by shaking, and incubate at 60°C for 60 minutes. Cool thoroughly on ice, remove the seal, and measure the absorbance at 570 nm. The amount of collagen components is calculated by comparing the absorbance of the sample with the standard.

[0050] The collagen component in the 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 in the cell structure distinguishable from other tissue components by a known staining method (e.g., immunostaining with an anti-collagen antibody or Masson's trichrome staining), and then calculating the ratio of the area in the entire cell structure where the collagen component is present, using macroscopic observation, various microscopes, image analysis software, etc. When defining by area ratio, there is no limitation on which cross section or surface in the cell structure defines the area ratio, but for example, when the cell structure is a sphere, it may be defined by a cross section passing through the approximate center.

[0051] For example, when the collagen component in the cell structure is defined by the area ratio, the area ratio is 0.01 to 99% based on the total area of ​​the cell structure, preferably 1 to 99%, preferably 5 to 90%, preferably 7 to 90%, preferably 20 to 90%, and more preferably 50 to 90%. The "collagen component in the cell structure" is as described above. The area ratio of the collagen component constituting the cell structure means the combined area ratio of the endogenous collagen component and the exogenous collagen component. The area ratio of the collagen component can be calculated, for example, by staining the obtained cell structure with Masson's trichrome and calculating the ratio of the area of ​​the collagen component stained blue to the total area of ​​the cross section passing through the approximate center of the cell structure.

[0052] The cell structure preferably has a survival rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more after trypsin treatment at a trypsin concentration of 0.25%, at a temperature of 37°C, pH 7.4, and for a reaction time of 15 minutes. Such cell structures are unlikely to be decomposed by enzymes during or after culture, and are stable. The survival rate can be calculated, for example, from the mass of the cell structure before and after trypsin treatment.

[0053] The cell structure may have a survival rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more after collagenase treatment at a collagenase concentration of 0.25%, at a temperature of 37° C., at pH 7.4, and for a reaction time of 15 minutes. Such a cell structure is unlikely to be decomposed by enzymes during or after culture, and is stable.

[0054] The thickness of the 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 laboratory animals and as a transplant material. The upper limit of the thickness of the cell structure is not particularly limited, and may be, for example, 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0055] Here, the "thickness of the cell structure" means the distance between both ends in the direction perpendicular to the main surface when the cell structure is sheet-shaped or rectangular. When the main surface has projections and recesses, the thickness means the distance at the thinnest part of the main surface.

[0056] Furthermore, when the cell structure is spherical or approximately spherical, the thickness means its diameter. Furthermore, when the cell structure is ellipsoidal or approximately ellipsoidal, the thickness means its minor axis. When the cell structure is approximately spherical or approximately ellipsoidal and has an uneven surface, the thickness means the shortest distance between two points where a line passing through the center of gravity of the cell structure intersects with the surface.

[0057] (Fibrin) The cell structure according to this embodiment may contain fibrin. Fibrin is a component that is generated when thrombin acts on fibrinogen to release A chains and B chains from the N-terminus of Aα chains and Bβ chains. Fibrin is a polymer and is generally insoluble in water. Fibrin is formed by contacting fibrinogen with thrombin.

[0058] [Method of manufacturing cell structures] The method for producing a cell structure having a vascular network between cells according to the present embodiment includes a contacting step of contacting cells with fragmented extracellular matrix components, and a culturing step of culturing the cells contacted with the fragmented extracellular matrix. In the contacting step, the cells (i) include at least adipocytes, stem cells, and vascular endothelial cells, or (ii) include at least adipocytes, stem cells, and vascular endothelial cells.

[0059] (contact process) In the production method according to this embodiment, the contacting step is a step of contacting fragmented extracellular matrix components with cells.

[0060] The cells in the contact step (i) include at least adipocytes, stem cells, and vascular endothelial cells, or (ii) include at least adipose stem cells and vascular endothelial cells. The cells and each cell are as described above. (i) may include cells other than adipocytes, stem cells, and vascular endothelial cells, and (ii) may include cells other than adipose stem cells and vascular endothelial cells. The stem cells in (i) are preferably adipose stem cells. Also, the adipocytes preferably include mature adipocytes.

[0061] By dispersing the fragmented extracellular matrix components in an aqueous medium, they become more easily accessible to cells in the aqueous medium, and can promote the formation of cell structures.

[0062] In the contacting step, the extracellular matrix components are contacted with the cells in an aqueous medium. Examples of the contacting step include, but are not limited to, a method of mixing an aqueous medium containing fragmented extracellular matrix components with an aqueous medium containing cells, a method of adding cells to an aqueous medium containing fragmented extracellular matrix components, a method of adding an aqueous medium containing extracellular matrix components to a culture solution containing cells, a method of adding cells to an aqueous medium containing extracellular matrix components, and a method of adding the extracellular matrix components and the cells to a previously prepared aqueous medium.

[0063] In addition, the order in which the cells are contacted with the fragmented extracellular matrix components is not particularly limited. For example, in the case of (i) above, the stem cells and vascular endothelial cells may be added to the aqueous medium containing the fragmented extracellular matrix components, and then the adipocytes may be added; the stem cells, vascular endothelial cells, and adipocytes may be added to the aqueous medium containing the fragmented extracellular matrix components in that order; the 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 the stem cells, vascular endothelial cells, and adipocytes. After each of the above additions, mixing may be performed by stirring or the like, or mixing may not be required. In addition, the contacting step may include a step of incubating for a certain period of time after contacting the fragmented extracellular matrix components with the cells.

[0064] The contacting step may be performed after forming a layer of cells in an aqueous medium. That is, the contacting step may be performed by forming a layer of cells in an aqueous medium and then contacting the extracellular matrix component. By forming a layer of cells before contacting the layer with the extracellular matrix component, a cell structure having a high cell density in the lower layer can be produced.

[0065] The fragmented extracellular matrix components can be obtained by the above-mentioned method. The fragmented extracellular matrix components may be obtained by fragmenting the extracellular matrix components in an aqueous medium. That is, the production method according to this embodiment may include a step of fragmenting the extracellular matrix components in an aqueous medium (fragmentation step) prior to 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 component may be any of those exemplified above, and may include a fragmented collagen component.

[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 may include a step of heating the extracellular matrix components after the fragmentation step and before the contacting step to crosslink at least a portion of the extracellular matrix components.

[0068] In the crosslinking step, the temperature (heating temperature) and time (heating time) when heating the extracellular matrix component can be appropriately determined. 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 appropriately set 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 step, heating may be performed in the absence of a solvent, or under reduced pressure conditions.

[0069] The production method according to this embodiment may include a drying step of drying the fragmented extracellular matrix components after the fragmentation step.

[0070] In the drying step, the defibrated extracellular matrix components are dried. Drying may be performed, for example, by freeze-drying. By performing the drying step after the defibration step, the aqueous medium is removed from a liquid containing the fragmented extracellular matrix components and the aqueous medium. Removal of the aqueous medium does not mean that no moisture is attached to the fragmented extracellular matrix components, but means that moisture is not attached to a degree that can be reasonably achieved by the above-mentioned general drying method.

[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, and may be 95% or less, 90% or less, 80% or less, or 75% or less, relative to the total number of cells in the contacting step.

[0072] The content of vascular endothelial cells may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more of the total number of cells in the contact step, and may be 95% or less, 90% or less, 80% or less, or 75% or less.

[0073] The concentration of the extracellular matrix components in the contact step can be appropriately determined depending on the shape and thickness of the desired cell structure, the size of the culture vessel, etc. For example, the concentration of the extracellular matrix components in the aqueous medium in the contact step may be 0.1 to 90% by mass, or 1 to 30% by mass.

[0074] The amount of the fragmented extracellular matrix components in the contact step is, for example, 1.0×10 6 For cells, the amount may be 0.1 to 100 mg, 0.5 to 50 mg, 0.8 to 25 mg, 1.0 to 10 mg, 1.0 to 5.0 mg, 1.0 to 2.0 mg, or 1.0 to 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 step, the mass ratio of the extracellular matrix components to the 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 the number of stem cells to vascular endothelial cells in the contact step (stem cells / vascular endothelial cells) 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 method may include adding fibrinogen and / or thrombin in the contact step, or after the contact step and before the culture step. When both fibrinogen and thrombin are added, for example, fibrinogen and thrombin may be added simultaneously, or fibrinogen may be added first and then thrombin may be added. The timing of adding fibrinogen and / or thrombin is not particularly limited, and for example, fibrinogen and / or thrombin may be added to an aqueous medium containing stem cells, vascular endothelial cells, adipocytes, and extracellular matrix components, or may be added to an aqueous medium containing stem cells, vascular endothelial cells, and extracellular matrix components. Also, for example, fibrinogen may be added to an aqueous medium containing stem cells, vascular endothelial cells, and extracellular matrix components, and then adipocytes may be added, and then thrombin may be added. By adding fibrinogen and / or thrombin, shrinkage that may occur in the culture step described below can be suppressed, and the shape and size of the cell structure can be easily controlled. In addition, since the suspension of cells and extracellular matrix components can be gelled, the suspension can be easily peeled off from the culture vessel after being dropped onto the culture vessel (support). Furthermore, when the cells contain mature adipocytes, the fat cells may float due to the influence of lipid droplets inside the mature adipocytes, resulting in the adipocytes being cultured in a non-uniform state relative to other cells. However, by gelling the suspension, it becomes easier to maintain a uniform mixture of the cells and extracellular matrix components and to maintain the cells and extracellular matrix components in close proximity to each other.

[0078] The method may further include a step of precipitating both the extracellular matrix components and the cells in the aqueous medium after the contact step and before the culture step. By carrying out such a step, the distribution of the extracellular matrix components and the cells in the cell structure becomes more uniform. There are no particular limitations on the specific method, but an example of the method is a method of centrifuging a culture solution containing the extracellular matrix components and the cells.

[0079] (Culture process) In the production method according to this embodiment, the culture step is a step of culturing cells in contact with the fragmented extracellular matrix.

[0080] The method for culturing the cells in contact with the fragmented extracellular matrix is ​​not particularly limited, and a suitable culture method can be used depending on the type of cells to be cultured. For example, the culture temperature may be 20°C to 40°C, or 30°C to 37°C. The pH of the 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 the cells in contact with the fragmented extracellular matrix is ​​not particularly limited, and may be, for example, a well insert, a low-adhesion plate, or a plate having a bottom shape such as U-shaped or V-shaped. The cells may be cultured while attached to the support, or may be cultured without being attached to the support, or may be cultured after being detached from the support during the culture. When culturing the cells without being attached to the support or culturing after being detached from the support during the culture, it is preferable to use a plate having a bottom shape such as U-shaped or V-shaped that inhibits adhesion of the cells to the support, or a low-adhesion plate.

[0082] The medium is not particularly limited, and a suitable medium can be selected depending on the type of cells to be cultured. Examples of the medium include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The medium may be a medium containing serum or a serum-free medium. The medium may be a mixed medium in which two types of media are mixed.

[0083] The cell density in the medium in the culture step can be appropriately determined depending on the shape and thickness of the desired cell structure, the size of the culture vessel, etc. For example, the cell density in the medium in the culture step can be set to 1 to 10 8 cells / mL, 3 ~10 7 The cell density in the medium in the culturing step may be the same as the cell density in the aqueous medium in the contacting step.

[0084] The cell structure produced by the production method according to this embodiment preferably has a shrinkage rate during culture of 20% or less, more preferably 15% or less, and even more preferably 10% or less. The 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 the first day after culture, and L3 represents the length of the corresponding part of the cell structure on the third day after culture. Shrinkage rate (%) = {(L1-L3) / L1} x 100

[0085] In the above example, the contraction rate is calculated from the cell structure on day 1 after culture and the cell tissue on day 3 after culture, but it may be calculated from the cell structure at any time point during the culture period, including the end of the culture. For example, it may be calculated from the cell structure on day 1 after culture and the cell structure on day 2 after culture, or it may be calculated from the cell structure on day 1 after culture and the cell structure on day 5 after culture, or it may be calculated from the cell structure on day 1 after culture and the cell structure on day 8 after culture.

[0086] After the above-mentioned culture step (hereinafter also referred to as the "first culture step"; the initial contact step is also referred to as the "first contact step"), a step of contacting the cells (second contact step) and a step of culturing the cells (second culture step) may be included. The cells in the second contact step and the second culture step may be the same type as the cells used in the first contact step and the first culture step, or may be a different type. A two-layered cell structure can be produced 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 produced, and tissue closer to a more complex living organism can also be produced.

[0087] According to the manufacturing method of this embodiment, it is possible to manufacture a cell structure having a vascular network between cells. The cell structure having a vascular network between cells is as described above.

[0088] The culture step may include culturing the cells in contact with the fragmented extracellular matrix in a state where they are not attached to a support. This makes it possible to produce a cell structure that is aggregated in a mass in a state where it is not attached to a support. When the cells in contact with the fragmented extracellular matrix are attached to a support, the culture step may include detaching the cells in contact with the fragmented extracellular matrix from the support. When the cells in contact with the fragmented extracellular matrix in the culture step are not attached to a support from the beginning, the cells are cultured as they are, making it possible to produce a cell structure that is aggregated in a mass in a state where it is not attached to a support.

[0089] The method for detaching the cells in contact with the fragmented extracellular matrix from the support is not particularly limited, and for example, a low-adhesive support may be used, and the cells may be detached from the support by adding a culture medium, the cells may be directly physically detached from the support using an instrument, or the cells may be detached from the support by applying vibration, or a support may be used whose surface is coated with a functional material that reacts to stimuli such as heat and light to release the bond between the support and the cells, and the cells may be detached from the support by applying a stimulus. When detaching the cells from the support by adding a culture medium, the medium exemplified above can be used as the culture medium.

[0090] In the culture step, examples of a method for culturing the cells in contact with the fragmented extracellular matrix in a state where they are not initially attached to a support include a method in which a suspension containing the cells in contact with the fragmented extracellular matrix is ​​gelled and gently dropped into a culture medium for culture, and a method in which the shape of the cells in contact with the fragmented extracellular matrix is ​​fixed to a certain 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 above culture step, the timing for detaching the cells in contact with the fragmented extracellular matrix from the support is not particularly limited, and may be, for example, 1 day to 7 days, 1 hour to 24 hours, 1 minute to 60 minutes, 5 minutes to 30 minutes, or 10 minutes to 20 minutes after the start of culture.

[0092] The culture period after the cells in contact with the fragmented extracellular matrix are detached from the support is not particularly limited, and may be one day or more, 1 to 21 days, 3 to 14 days, or 7 to 14 days.

[0093] [Cell tissue and its manufacturing method] By using a plurality of the above-mentioned cell structures assembled in a mass without being attached to a support, i.e., "cell structures comprising fragmented extracellular matrix components and cells, having a vascular network between the cells, and assembled in a mass without being attached to a support, wherein the cells include at least fat cells and vascular endothelial cells," a cellular tissue in which the vascular network is connected between the plurality of cell structures can be produced.

[0094] The production of the cell tissue includes subjecting the cell structures not attached to a plurality of supports to suspension culture. The plurality of cell structures are attached to each other during the suspension culture process, and the vascular networks are connected between the cell structures, so that a large cell tissue with a connected vascular network can be easily produced. The number and size of the cell structures used can be appropriately selected depending on the application of the cell tissue, the desired size of the cell tissue, and the like. In addition, the type of medium (culture solution) and culture conditions used for suspension culture can also be appropriately selected, and 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 the present embodiment has a vascular network formed between cells like living tissue, and is expected to be easily engrafted when transplanted into animals such as mammals, and therefore can be used for transplantation. The cell structure used for transplantation may be one or more. When there are more than one cell structure, for example, 1 to 1000, 10 to 500, or 50 to 200 can be used.

[0096] The animal to be transplanted is not particularly limited, and may be, for example, a mammal, a human, or a non-human animal such as a monkey, a dog, a cat, a rabbit, a pig, a cow, a mouse, or a rat.

[0097] The transplantation method according to the present embodiment includes transplanting the cell structure having the vascular structure according to the present embodiment into an animal. Before the transplantation, the method may further include preparing an animal to be transplanted and / or preparing a cell structure by the above-mentioned method. The transplantation method is not particularly limited, and may be performed by a known surgical method or the like as appropriate according to the transplantation target, etc. Examples of the surgical method include incising the skin of the transplantation target and transplanting directly under the skin, or injecting under the skin of the transplantation target with a syringe or the like. The cell structure to be transplanted may be one or more, or may be a cell tissue containing multiple cell structures. The cell structure or cell tissue may be collected from a culture medium, or may be a cell structure or cell tissue that has been appropriately gelled or semi-gelled (e.g., fibrin gel) according to the form of transplantation, or a dispersion liquid in which multiple cell structures are dispersed may be used. In addition, fibrin may be added to a cell structure collected from a culture medium and aggregated in a mass form without being attached to multiple supports, and the cell structure may be used for transplantation. The cell structure according to this embodiment containing adipocytes can be applied to tissue reconstruction after, for example, trauma, soft tissue defects caused by tumor removal, and mastectomy.

[0098] The vascular network of the transplanted cell structure is connected to the transplant subject's own blood vessels around the transplant site. When multiple cell structures assembled in a mass without being attached to a support are used, the vascular network is also connected between the multiple cell structures. The adipose tissue formed in the transplant subject by transplanting multiple cell structures assembled in a mass without being attached to a support has a vascular network connected between the multiple cell structures and also connected to the transplant subject's own blood vessels, and therefore has better adhesion.

[0099] The method for producing a non-human model animal according to the present embodiment includes transplanting the cell structure according to the present embodiment into a non-human animal. Before the transplantation, the method may further include preparing a non-human animal to be transplanted and / or preparing a cell structure by the above-mentioned method. The transplantation method is as described above, and the cell structure to be transplanted may be one or more, or may be a cell tissue containing multiple cell structures. The method for producing a non-human model animal according to the present embodiment may include growing the cell structure for, for example, 7 days or more, 30 days or more, or 90 days or more after transplanting the cell structure into the non-human animal. The non-human model animal according to the present embodiment can be used to apply data obtained using the cell structure in animal experiments to humans. In order to produce a non-human model animal, it is preferable to use a non-human animal in which rejection of a transplant (graft) is suppressed, for example, a non-human animal with reduced or impaired immunity. The non-human model animal can be used, for example, as a pathological in vitro model of 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 of this embodiment itself can be used as a substitute for experimental animals, transplant materials, etc., and specific examples of its application include tissue reconstruction, pathological in vitro models, pharmaceutical screening (drug evaluation), and cosmetic assay screening, as described above. EXAMPLES

[0101] <Test Example 1: Preparation of defibrated collagen component> A collagen component in which at least a part is cross-linked (cross-linked collagen component) was obtained by heating 100 mg of pig skin-derived collagen type I sponge fragments (manufactured by Nippon Ham Co., Ltd.) at 200°C for 24 hours. No significant change in the appearance of the collagen was observed 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 defibrated by homogenizing for 6 minutes using a homogenizer (As One VH-10).

[0102] The mixture was centrifuged at 10,000 rpm for 10 minutes at 21°C. 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 ultrasonicated at 100 V for 20 seconds 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 ultrasonic treatments, the collagen solution was filtered through a filter with a pore size of 40 μm 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 dry body. The average length of the sCMF was 14.8 ± 8.2 μm (N = 20).

[0103] <Test Example 2: Preparation and evaluation of cell structures (1)> The cells, reagents and preparation methods used in preparing the cell structures are as follows. (cells and collagen) Human adipose tissue (from thigh) for obtaining primary human mature adipocytes and human adipose stem cells (ADSC) (provided by Kyoto Prefectural University of Medicine Hospital) Human umbilical vein-derived endothelial cells (HUVEC) (Lonza #C-2517A) Defibrillated collagen component (sCMF) (prepared in Test 1)

[0104] (reagent) Bovine pancreatic insulin (Sigma #I1882) · Bovine plasma-derived thrombin lyophilized powder (Sigma #T4648) Collagenase type I from Clostridium histolyticum (Sigma #C0130) Bovine plasma-derived fibrinogen type IS (Sigma #F8630) DMEM (high glucose, Nacalai Tesque) EGM-2MV BulletKit with growth factors (Lonza #C-2517A)

[0105] (Culture medium and various solutions) EGM-2 medium: 500mL of EBM-2 mixed with EGM-2 supplement growth factors and stored at 4℃ 10mg / mL insulin stock solution: 100mg of the bovine pancreatic insulin described above was dissolved in 10mL of 1% glacial acetic acid solution (pH ≦2) diluted with water, and the solution was dispensed in equal amounts into Eppendorf tubes and stored at -20℃. 2mg / mL collagenase solution: Mix 2.5g of BSA with 50mL of DMEM (0% FBS, 1% antibiotics). To digest all the adipose tissue in a 6-well plate, mix 26mg of collagenase type I with 13mL of DMEM (0% FBS, 5% BSA, 1% antibiotics) and filter through a 0.2μm filter. 50mg / mL fibrinogen stock solution: Weigh 50mg of fibrinogen into an Eppendorf tube and immediately add 1mL of DMEM (0% FBS, 1% antibiotics). After manually mixing by shaking the tube, place in a 37℃ water bath for 3-5 minutes, filter through a 0.2μm filter, and dispense equal amounts into Eppendorf tubes for use. 202U / mL thrombin stock solution: Measure 202U of thrombin into an Eppendorf tube, immediately add 1mL of DMEM (0% FBS, 1% antibiotics), and dissolve in a 37℃ water bath for 3-5 minutes. Then, filter through a 0.2μm filter and dispense equal amounts into Eppendorf tubes for use.

[0106] (Production method) Human adipose tissue fragments were washed with PBS containing 5% antibiotics. 4-6 g of tissue was divided into 6 wells of a 6-well plate. The tissue was minced into small pieces of approximately 1-3 mm in size using scissors and tweezers in 2 mL of 2 mg / mL collagenase solution. After incubation at 37°C and 230 rpm for 1 h, the tissue was mixed with a 10 mL pipette for 30 min. The lysate was filtered through an iron mesh filter with a pore size of 500 μm, and 2 mL of DMEM was added per well to recover all the digested cells, followed by centrifugation at 200 g for 3 min at room temperature (15-25°C). Mature adipocytes were found in the upper yellow oily layer, while adipose stem cells and blood cells were found in the pellet. Using a long needle and a 10 mL syringe, the medium between the upper and lower layers was aspirated and discarded, and 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% antibiotics). When washing, the cells were centrifuged as described above to separate them into three layers: an upper layer, a lower layer, and the medium between the upper and lower layers, and the medium between the upper and lower layers was aspirated and discarded. After washing twice, the cells were washed with 25 mL of DMEM.

[0107] Only the upper layer containing mature adipocytes was collected and dispensed into an Eppendorf tube. Nuclei were stained with Hoechst stain (1000-fold diluted Hoechst, stained for 15 minutes), and the number of cells was counted using a Turker Burk hemocytometer under a fluorescent microscope.

[0108] The pellet containing ADSCs was suspended in 10 mL of DMEM and seeded in a 10 cm dish for subculture. The ADSCs were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and the cell number was counted.

[0109] HUVEC purchased from Lonza were suspended in 10 mL of DMEM, seeded in a 10 cm dish, and subcultured. HUVEC were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and the cell number was counted.

[0110] 1 mg of sCMF was weighed out, 100 μL of DMEM was added, and mixed gently until only small particles of sCMF were observed. The mixture was centrifuged at 10,000 rpm at room temperature for 1 minute, the supernatant was aspirated, and an sCMF pellet was obtained. 250,000 cells of ADSC and 125,000 cells of HUVEC (ADSC:HUVEC=2:1) ​​were gently added onto the sCMF pellet, and the mixture was centrifuged at 3,500 rpm at room temperature for 1 minute without mixing, 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 further added and gently mixed. A small amount of DMEM was added as necessary to adjust the total volume to 70 μL. 0.15 U of thrombin (0.71 μL of 202 U / mL thrombin stock solution) was immediately added and mixed, after which the mixture was slowly seeded into a transwell placed on a 6-well adaptor on a 6-well plate.

[0111] The cells were incubated in a 37°C incubator for 1 hour to gel, and 12 mL of EGM-2 medium containing insulin at a final concentration of 10 μg / mL was added. 12 mL of medium was replaced every 2 to 3 days until the 7th day of culture.

[0112] Fluorescence imaging of the cell structures was performed as follows. The transwell containing the obtained cell structures was 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 tissue was 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 inside the transwell, 500 μL outside the transwell) and washed three times with 2 mL of PBS.

[0113] The tissue was blocked with 1% BSA / PBS solution for 1 hour at room temperature (500 μL in the transwell, 500 μL outside the transwell). The BSA solution was aspirated, and 100 μL of the primary antibody solution (CD31 and perilipin diluted 100-fold with 1% BSA / PBS solution) was added (50 μL in the transwell, 50 μL outside the transwell). A wet tissue 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 the secondary antibody solution (AlexaFluor647-labeled anti-mouse anti-CD31 antibody and AlexaFluor488-labeled anti-rabbit anti-perilipin antibody diluted 200-fold with 1% BSA / PBS solution, Hoechst diluted 1000-fold) was added (50 μL in the transwell, 50 μL outside the transwell). A wet tissue was placed under the plate, which was then covered with aluminum foil and incubated at room temperature for 2 hours, after which the plate was 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 observed using a confocal laser scanning microscope (FV3000, Olympus Corporation) with laser excitation light of 640 nm (AlexaFluor 647) and 488 nm (AlexaFluor 488).

[0115] The diameter of lipid droplets in the cell structures was measured using an electron microscope. The diameter of blood vessels and the length between blood vessel branches were measured using Image J.

[0116] Figure 1 shows the results of fluorescence observation of a cell structure with a vascular network (magnified 20 times). (a) is a living tissue in which adipose tissue taken from a living body was fixed as is, and (b) is a cell structure. In the cell structure, it was confirmed that a vascular network was formed surrounding mature adipocytes (large round, single-eye-shaped lipid droplets) as in living tissue. These vascular networks were formed toward the inside of the cell 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)). In addition, the formed blood vessels were hollow like living tissue, and both thick ones (e.g., 10 μm or more and less than 25 μm) and thin ones (e.g., more than 0 μm and less than 10 μm) were observed, like living tissue. As shown in Figure 2, it was found that the number of blood vessel branches was also close to the number in living tissue. In addition, the distribution of the length between blood vessel branches was 0-100μm: 42.5%, 100-200μm: 39.4%, 200μm or more: 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 biological tissues (0-100μm: 61.8%, 100-200μm: 24.7%, 200μm or more: 13.5% (N=180)). In Test Example 2, the length between blood vessel branches was often 50μm-100μm, which is close to the size of mature adipocytes. For example, as described 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 blood vessels surround individual fat cells in adipose tissue in vivo, and blood vessels are formed between fat cells. The distribution of the length between the branches of blood vessels as described above suggests the possibility that the cell structure of Test Example 2 can faithfully mimic actual adipose tissue in vivo.

[0117] <Test Example 3: Preparation and evaluation of cell structures (2)> A cell structure was produced in the same manner as in Test Example 2, except that 500,000 mature adipocytes, 2 mg of sCMF, and 0.6 mg of fibrinogen were used, and a cell structure in which a vascular network was formed surrounding the mature adipocytes was produced. Figure 3 shows the results of fluorescent observation (10x magnification) of a cell structure stained in the same manner as in Test Example 2. It was shown that a cell structure with a vascular network could be produced even if the number of mature adipocytes and the amount of sCMF were changed.

[0118] <Test Example 4: Preparation and evaluation of cell structures (3)> A cell structure was produced in the same manner as in Test Example 2, except that no mature adipocytes were used, and 2 mg of sCMF, 0.6 mg of fibrinogen, and 0.3 U of thrombin were used. A cell structure with a vascular network was produced. Figure 4 shows the results of fluorescent observation (4x magnification) of a cell structure in which only the blood vessels were stained with an anti-CD31 antibody. It was shown that a cell structure with a vascular network can be produced without using mature adipocytes.

[0119] <Test Example 5: Preparation and evaluation of cell structures (4)> A cell structure was prepared in the same manner as in Test Example 2, except that no mature adipocytes were 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. A cell structure with a vascular network was prepared. Figure 5 shows the results of fluorescent observation of a cell structure in which only the blood vessels were stained using an anti-CD31 antibody (magnified 4 times). It was shown that a cell structure with a vascular network could be prepared even if the ratio of ADSC and HUVEC was changed without using mature adipocytes.

[0120] <Test Example 6: Preparation and evaluation of cell structures (5)> Instead of mature adipocytes, ADSCs, and HUVECs, adipose tissue obtained by liposuction from human thighs (biological tissue) was used, and the total amount before seeding was adjusted to 60 μL. Adipose tissue was prepared by finely chopping 3 g of collected biological tissue into approximately 1 to 3 mm pieces using scissors and tweezers, and slowly pipetting several times using a 10 mL syringe to make it liquid. 60 μL was taken from the liquid adipose tissue and mixed with sCMF. The adipose tissue obtained by liposuction contains mature adipocytes, adipose stem cells, and vascular endothelial cells. Figure 6 shows the results of fluorescent observation (10 times magnification) of a cell structure stained by the same method as in Test Example 2. It was shown that a cell structure having a vascular network can be prepared even if adipose tissue obtained from a biological tissue is used instead of mature adipocytes, ADSCs, and HUVECs. It was confirmed that a cell structure having a vascular network can also be prepared when 2 mg of sCMF is used.

[0121] In all of Test Examples 2 to 6, it was confirmed that a cell structure having a vascular network could be produced, but when Test Examples 2 to 6 were compared, it was found that the use of 1 mg of sCMF tended to produce a cell structure having a vascular network more similar to that of living tissue than the use of 2 mg of sCMF. In addition, the ratio of ADSC to HUVEC was ADSC:HUVEC=2:1, which tended to produce a cell structure having a vascular network more similar to that of living tissue than the use of ADSC:HUVEC=5:1.

[0122] <Comparative Example> A cell structure was prepared in the same manner 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 structure. In addition, a cell structure was prepared in the same manner as in Test Example 2, except that no ADSC was included. Only a very small amount of blood vessel formation was observed in the prepared cell structure, and no formation of a vascular network surrounding mature adipocytes was confirmed.

[0123] <Test Example 7: Preparation and evaluation of cell structure (6)> Cells, reagents, culture medium, and various solutions used in the production of cell structures were prepared in the same manner as in Test Example 2. The defibrated collagen component (sCMF) used was also prepared in the same manner as in Test Example 1. An overview of this test example is shown in Figure 7. In Figure 7, the circles in the droplet on the left represent mature adipocytes, the white diamonds represent ADSCs, and the short grey bars represent HUVECs.

[0124] (Production method) Human adipose tissue fragments were washed with PBS containing 5% antibiotics. 4-6 g of tissue was divided into 6 wells of a 6-well plate. The tissue was minced into small pieces of approximately 1-3 mm in size using scissors and tweezers in 2 mL of 2 mg / mL collagenase solution. After incubation at 37°C and 230 rpm for 1 h, the tissue was mixed with a 10 mL pipette for 30 min. The lysate was filtered through an iron mesh filter with a pore size of 500 μm, and 2 mL of DMEM was added per well to recover all the digested cells, followed by centrifugation at 200 g for 3 min at room temperature (15-25°C). Mature adipocytes were found in the upper yellow oily layer, while adipose stem cells and blood cells were found in the pellet. Using a long needle and a 10 mL syringe, the medium between the upper and lower layers was aspirated and discarded, and 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% antibiotics). When washing, the cells were centrifuged as described above to separate them into three layers: an upper layer, a lower layer, and the medium between the upper and lower layers, and the medium between the upper and lower layers was aspirated and discarded. After washing twice with 25 mL of PBS (5% BSA, 1% antibiotics), the cells were washed with 25 mL of DMEM.

[0125] Only the upper layer containing mature adipocytes was collected and dispensed into an Eppendorf tube. 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 fluorescent microscope.

[0126] The pellet containing ADSCs was suspended in 10 mL of DMEM and seeded in a 10 cm dish for subculture. The ADSCs were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and the cell number was counted.

[0127] HUVEC purchased from Lonza were suspended in 10 mL of DMEM, seeded in a 10 cm dish, and subcultured. HUVEC were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and the cell number was counted.

[0128] To obtain one roughly spherical cell structure (hereinafter 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 mixed gently until only small particles of sCMF were observed. The mixture was centrifuged at 10,000 rpm at room temperature for 1 minute, the supernatant was aspirated, and an sCMF pellet was obtained. 220,000 cells of ADSCs and 275,000 cells of HUVECs were gently added onto the sCMF pellet, the mixture was centrifuged at 3,500 rpm at room temperature for 1 minute, and the supernatant was aspirated. Fibrinogen (32 μL of 50 mg / mL fibrinogen stock solution) was added and gently mixed with the cells and sCMF. 650,000 cells of mature adipocytes were further added and gently mixed. A small amount of DMEM was added to adjust the total volume to 200 μL. Thrombin (3.9 μL of 202 U / mL thrombin stock solution) was immediately added and mixed briefly, after which the mixture (enough for 40 cell balls) was seeded onto a low-attachment 96-well plate (IWAKI #4860-800LP) at 5 μL per well (enough for one cell ball).

[0130] The mixture was incubated in a 37° C. incubator for 15 minutes to allow it to gel, and 300 μL of EGM-2 medium containing insulin at a final concentration of 10 μg / mL was added.

[0131] After 24 hours of culture, 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 replaced every 2 to 3 days until the 7th day of culture.

[0132] Fluorescence imaging of the cell structures was performed as follows. The transwell containing the obtained cell structures was 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. The tissue was washed three times with 200 μL of PBS. For immunostaining, the cells were permeabilized with 200 μL of 0.05% Triton / PBS at room temperature for 7 minutes and washed three times with 200 μL of PBS.

[0133] The tissue was blocked with 200 μL of 1% BSA / PBS solution at room temperature for 1 hour. The BSA solution was aspirated, and 100 μL of primary antibody solution (CD31 and perilipin diluted 100-fold with 1% BSA / PBS solution) was added. A wet tissue was placed under the plate, covered with aluminum foil, and incubated overnight at 4°C. After washing three times with 200 μL of PBS, a secondary antibody solution (AlexaFluor647-labeled anti-mouse anti-CD31 antibody and AlexaFluor488-labeled anti-rabbit anti-perilipin antibody diluted 200-fold with 1% BSA / PBS solution, Hoechst diluted 1000-fold) was added. A wet tissue was placed under the plate, covered with aluminum foil, and incubated at room temperature for 2 hours, and then washed four times with 200 μL of PBS.

[0134] The cell balls were placed directly on the complete plate of a confocal quantitative image cytometer CQ1 (Yokogawa Electric Corporation), and the stained cell structures were observed using the above-mentioned confocal quantitative image cytometer CQ1 with laser excitation light of 640 nm (AlexaFluor 647) and 488 nm (AlexaFluor 488).

[0135] Figure 8 shows the results of fluorescent observation of cell balls with vascular networks by Nile red staining and CD31 staining. CD31 staining was performed in the same manner as in Test Example 2, and Nile red staining was performed by the usual method. The photograph on the right is a further enlargement of one of the cell balls on the left. It was confirmed that in the cell balls, vascular networks were formed surrounding mature fat cells (large round, single-lens shaped fat droplets) like in living tissue. These vascular networks were formed deep inside the cell balls.

[0136] Figure 9 shows the results of observing a section of a cell ball with a vascular network by CD31 immunohistochemical staining. From this result, many lumens were observed in the cell ball, and it was confirmed that the vascular network was formed deep inside the cell structure.

[0137] Figure 10 shows the average diameter (n=12 cell balls / volume) of the cell balls produced by the method of this test example after 7 days of culture. The average diameter of the 5μL cell balls was 1256μm, and the average diameter of the 10μL cell balls was 1857μm.

[0138] <Test Example 8: Preparation and evaluation of cell tissue containing multiple cell balls> Multiple 5 μL cell balls prepared in Test Example 7 after 7 days of culture were placed in contact with each other and cultured in suspension in 10 mL of culture medium for 7 days. The cells were stained and observed in the same manner as in Test Example 7. As a result, as shown in FIG. 11, multiple cell balls (five in FIG. 11) aggregated and united to obtain a cellular tissue. It was confirmed that the cellular tissue formed by the united five cell balls had a vascular network connected not only within each cell ball, but also between the multiple cell balls (left and right of FIG. 11).

[0139] <Test Example 9: Cell ball transplantation test and evaluation> Six immunodeficient mice were prepared, and their back skin was incised. The following (1) to (3) were injected into the incision, followed by suturing. Three mice were grown for 30 days and the other three for 90 days. (1) A mixture of 111 cell balls after culture prepared in Test Example 7 suspended in 100 μL of a solution containing 2.5 mg of fibrinogen and 1.25 U of thrombin. (2) A mixture of 111 cell balls prepared in the same manner as in (1) except that HUVECs were not used, suspended in 100 μL of a solution containing 2.5 mg fibrinogen and 1.25 U thrombin. (3) In Test Example 6, a cell structure (100 μL) was prepared using adipose tissue obtained by liposuction from a human thigh (biological tissue).

[0140] After 30 days of growth, tissue was collected from the transplant site of each individual. Adipose tissue was formed at the site where (2) above was transplanted, but no vascular network was observed. Adipose tissue was formed at the site where (3) above was transplanted, and although a slight vascular network was observed on the tissue surface, the presence of large oil droplets was also observed. The formation of oil droplets indicates the death of fat cells. On the other hand, at the site where (1) above, which used the cell ball prepared in Test Example 7, was transplanted, the formation of adipose tissue with a vascular network spread throughout the tissue surface was observed. Furthermore, no oil droplets were observed in the tissue at the site where (1) above was transplanted, indicating better adhesion after transplantation.

[0141] After 30 days of growth, tissue was collected from the transplantation site (1) above of the individual, and perilipin staining and DAPI staining were performed in the same manner as in Test Example 2 (Figure 12). DAPI staining was performed by a conventional method. A: SFT indicates tissue collected from the transplantation site (3) above, and C: 3DVFT indicates tissue collected from the transplantation site (1) above. The upper row of Figure 12 shows the results of observation in a bright field, the middle row shows the results of perilipin staining, and the lower row shows the results of DAPI staining. It was confirmed that mature adipocytes were formed in the tissue formed at the transplantation site.

[0142] In addition, tissue was collected from the transplantation site (1) of the individual after 90 days of growth, and stained with perilipin and CD31 in the same manner as in Test Example 2 (FIG. 13). The upper row of FIG. 13 shows the results of observation in a bright field, the middle row shows the results of CD31 staining, and the lower row shows the results of DAPI staining. It was confirmed that not only were mature adipocytes formed in the tissue formed at the transplantation site, but also that a vascular network was formed.

[0143] From the above, it was demonstrated that the cell structure according to this embodiment has excellent adhesion after transplantation and is suitable for transplantation.

Claims

1. comprising a plurality of cell structures, The cell structure is a gelled cell structure comprising fragmented extracellular matrix components and cells, having a vascular network between the cells, and assembled in a clump without being attached to a support, wherein the cells include at least adipocytes and vascular endothelial cells. The average length of the fragmented extracellular matrix components is between 100 nm and 400 μm. A cellular tissue in which the vascular network is connected between the plurality of cellular structures.

2. The cell tissue according to claim 1, wherein the fragmented extracellular matrix component comprises a fragmented collagen component.

3. The cell tissue according to claim 2, wherein the fragmented collagen component is homogenized fragmented collagen.

4. The cell tissue according to any one of claims 1 to 3, wherein the content of extracellular matrix components in the cell structure is 0.01 to 90% by mass, based on the dry weight of the cell structure.

5. The cell tissue according to any one of claims 1 to 4, wherein the cell structure comprises fibrin.

6. A cell tissue according to any one of claims 1 to 5, for transplantation.

7. A method for producing cell tissue, This includes suspension culture of multiple cell structures. The cell structure is a gelled cell structure comprising fragmented extracellular matrix components and cells, having a vascular network between the cells, and assembled in a clump without being attached to a support, wherein the cells include at least adipocytes and vascular endothelial cells. The average length of the fragmented extracellular matrix components is between 100 nm and 400 μm. A method for producing the cell tissue, wherein the vascular network is connected between the plurality of cell structures.

8. The method includes obtaining the cell structures before performing suspension culture of the plurality of cell structures, Obtaining the cell structure is 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. The process includes a culture step of culturing cells that have come into contact with fragmented extracellular matrix components, The manufacturing method according to claim 7, wherein the culture step includes culturing cells that have come into contact with the fragmented extracellular matrix components in a state in which they do not adhere to a support.

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

10. The manufacturing method according to claim 9, wherein the fragmented collagen component is homogenized fragmented collagen.

11. The manufacturing method according to any one of claims 7 to 10, wherein the content of the extracellular matrix component in the cell structure is 0.01 to 90% by mass based on the dry weight of the cell structure.

12. The manufacturing method according to any one of claims 7 to 11, wherein the cell structure comprises fibrin.

13. The manufacturing method according to any one of claims 7 to 12, wherein the cell tissue is for transplantation.

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