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, and 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] Known techniques for artificially creating structures that mimic biological tissue include a method for producing a three-dimensional tissue (Patent Document 1), which involves three-dimensionally arranging cells coated with a collagen-containing membrane to form a three-dimensional tissue, and a method for producing a three-dimensional cellular tissue (Patent Document 2), which involves mixing cells with a cationic substance and an extracellular matrix component to obtain a mixture, collecting the cells from the mixture, and forming a cell aggregate on a substrate. The present inventors have also proposed a method for producing a large three-dimensional tissue (1 mm or more thick) using a relatively small number of cells by contacting cells with fragmented exogenous collagen (Patent Document 3). These three-dimensional tissues are expected to be used as substitutes for laboratory animals, transplant materials, and the like. [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] The above-described method for producing a three-dimensional tissue structure allows for the creation of a thick three-dimensional tissue structure. However, no method for producing adipose tissue, which has a vascular network formed between cells like in living tissue, was 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 treating a fibroblast cell comprising: A cell structure having a vascular network between cells, A 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 components 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% by 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 cells with fragmented extracellular matrix components, wherein the cells (i) contain at least adipocytes, stem cells, and vascular endothelial cells, or (ii) contain at least adipocytes, stem cells, and vascular endothelial cells; a culturing 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 treatment agent is 0.1 to 100 mg per 1000 cells.

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

[12] , wherein the ratio of the number of stem cells to the number of 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 to 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 treating a fibroblast cell 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, A 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 fragmented extracellular matrix components with cells, wherein the cells (i) contain at least adipocytes, stem cells, and vascular endothelial cells, or (ii) contain at least adipocytes, stem cells, and vascular endothelial cells; a culturing 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 culturing step comprises culturing the cells in contact with the fragmented extracellular matrix in a state where the cells are not attached 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 the 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 it 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 it for 90 days or more.

[28] A method for evaluating the effect of a drug that inhibits or promotes adipose tissue metabolism, using the cell structure according to any one of [1] to [8],

[19] and

[20] .

[29] An administration step of administering a drug that inhibits or promotes adipose tissue metabolism to the cell structure; an evaluation step of evaluating the effect of the drug based on metabolic changes in the cell structure to which the drug is administered; The method according to

[28] , comprising:

[30] The evaluation step Altered glucose and / or fatty acid uptake, and / or Changes in the release of incorporated glucose and / or fatty acids The method according to

[29] , comprising evaluating the above using as an indicator.

[31] A method for screening for a drug that inhibits or promotes adipose tissue metabolism, using the cell structure according to any one of [1] to [8],

[19] and

[20] .

[32] Measuring metabolism in a cell structure to which a drug has been administered; A step of comparing the metabolism in the cell structure to which the drug has been administered with the metabolism in the cell structure to which the drug has not been administered, and selecting the drug as a candidate drug for inhibiting adipose tissue metabolism if the metabolism in the cell structure to which the drug has been administered is lower, or Measuring metabolism in the cell structure to which the drug has been administered; The method described in

[31] , comprising the steps of comparing the metabolism in a cell structure administered with a drug with the metabolism in a cell structure not administered with a drug, and if the metabolism in the cell structure administered with a drug is higher, selecting the drug as a candidate drug for promoting the metabolism of adipose tissue.

[33] The method according to

[32] , wherein the comparison of metabolism in the cell structure is carried out using the uptake of glucose and / or fatty acids and / or the release of the taken-up glucose and / or fatty acids as indicators. [Effects of the Invention]

[0007] According to the present invention, a cell structure having a vascular network between cells can be easily produced. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows photographs showing the results of observation of (a) biological tissue and (b) the cell structure of Test Example 2 by perilipin staining and CD31 staining. [Figure 2] 10 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. [Figure 3] 1 shows photographs showing the results of observing the cell structure of Test Example 3 by perilipin staining and CD31 staining. [Figure 4]1 shows photographs showing the results of observing the cell structure of Test Example 4 by CD31 staining. [Figure 5] 1 shows photographs showing the results of observing the cell structure of Test Example 5 by CD31 staining. [Figure 6] 1 shows photographs showing the results of observing the cell structure of Test Example 6 by perilipin staining and CD31 staining. [Figure 7] 1 is a diagram showing an overview of Test Example 7. The circles in the droplet on the left represent mature adipocytes, the white diamonds represent ADSCs, and the short gray bars represent HUVECs. [Figure 8] 10 is a photograph showing the results of fluorescent observation of cell balls having a vascular network in Test Example 7, using Nile Red staining and CD31 staining. (b) is a further enlarged view of one of the cell balls in (a). [Figure 9] 10 shows photographs showing the results of observing cell balls with vascular networks using CD31 staining. [Figure 10] 1 is a graph showing the average diameter (n=12 cell balls / amount) of cell balls prepared 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 ((a) and (b) are partially enlarged photographs), and (c) is a photograph of aggregated cell balls on a plate observed in bright field. [Figure 12] 12 shows photographs showing the results of fluorescent observation, by perilipin staining and DAPI staining, of tissue collected 30 days after transplantation in Test Example 9. A: SFT indicates tissue collected from the site where adipose tissue obtained by liposuction from a human thigh (living tissue) was transplanted, and C: 3DVFT indicates tissue collected from the site where the cell ball of Test Example 9 (1) was transplanted. The top row of Figure 12 shows the results of bright field observation, the middle row shows the results of perilipin staining, and the bottom row shows the results of DAPI staining. [Figure 13]13 shows photographs showing the results of fluorescent observation of tissues collected 90 days after transplantation in Test Example 9, using CD31 staining and DAPI staining. A: SFT shows tissue collected from the site where adipose tissue obtained by liposuction from a human thigh (living tissue) was transplanted, and C: 3DVFT shows tissue collected from the site where the cell ball of Test Example 9 (1) was transplanted. The top row of Figure 13 shows the results of bright field observation, the middle row shows the results of CD31 staining, and the bottom row shows the results of DAPI staining. [Figure 14] Photographs showing the results of fluorescence observation of the cell structures at 10, 30, 60, 120, and 150 minutes after incubation. [Figure 15] The figure shows a comparison of fluorescence intensity after 60 minutes in a cell structure containing vascular endothelial cells (b) and a tissue structure without vascular endothelial cells (a) on days 7 and 14 of formation. Col I indicates the use of sCMF made with type I collagen, and Col I+III indicates the use of sCMF made with a mixture of types I and III collagen. [Figure 16] The comparison of fluorescence intensity is shown at 20, 45, 70, 90, and 135 minutes after incubation of the endothelial cell-free tissue structure. [Figure 17] Graph (a) shows a comparison of fluorescence intensity (amount of glucose uptake) at 20, 45, 70, and 90 minutes after incubation of a tissue not containing vascular endothelial cells in Test Example 11. Graph (b) shows a comparison of fluorescence intensity (amount of fatty acid uptake) at 5, 30, and 60 minutes after incubation of a tissue not containing vascular endothelial cells in Test Example 12. [Figure 18] The figure shows a comparison of the fluorescence intensity of a cell structure containing vascular endothelial cells (b) and a tissue structure not containing vascular endothelial cells (a) at 0, 5, 30, and 60 minutes after incubation. [Figure 19] A comparison of the amount of glucose released (a) and the amount of fatty acid released (b) after the second incubation is shown. DETAILED DESCRIPTION OF THE INVENTION

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

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

[0011] It is difficult to maintain an artificially created, thick, three-dimensional tissue without blood vessels, and it is believed that oxygen and other substances must be supplied from the outside. In contrast, the cell structure according to this embodiment has a vascular network formed between the cells, similar to living tissue, and is therefore expected to be able to be maintained for a long period of time. It is also expected to be more likely to take root when transplanted into mammals, etc.

[0012] As used herein, the term "cell structure" refers to an aggregate (agglomerated cell mass) of cells arranged three-dimensionally via extracellular matrix components, artificially produced by cell culture. The shape of the cell structure is not particularly limited, and examples include sheet-like, spherical, approximately spherical, ellipsoidal, approximately ellipsoidal, hemispherical, approximately hemispherical, semicircular, approximately semicircular, rectangular, and approximately rectangular. Biological tissues include sweat glands, lymphatic vessels, sebaceous glands, and the like, and have a more complex structure than cell structures. Therefore, cell structures and biological tissues are easily distinguishable. Furthermore, cell structures may be aggregated in a mass state adhered to a support, or may be aggregated in a mass state not adhered to a support. By using multiple cell structures aggregated in a mass state not adhered to a support, it is possible to efficiently produce cellular tissues in which a vascular network is connected between the multiple cell structures, 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 the cell may be a somatic cell derived from bone, muscle, internal organs, nerve, brain, bone, skin, blood, or the like, or 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 with self-renewal and pluripotency. Stem cells include pluripotent stem cells, which have the ability to differentiate into any cell type, and tissue stem cells (also called somatic stem cells), which have the ability to differentiate 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] As used herein, "adipocytes" refers to all adipocytes excluding adipose stem cells. Adipocytes include mature adipocytes and adipocytes not included in adipose stem cells. Preferably, the adipocytes include mature adipocytes, more preferably 90% or more of the total number of adipocytes are mature adipocytes, and even more preferably all are mature adipocytes. Adipocytes may be cells collected from, for example, subcutaneous adipose tissue and epicardial-derived adipose tissue, or collected cells (e.g., adipose stem cells) may be induced to differentiate and used. The adipocytes are not particularly limited, but when adipose tissue constructed from adipocytes is ultimately used to resemble tissue in a specific location in the body, it is preferable to use those derived from tissue corresponding to the tissue in that location.

[0017] The size of lipid droplets can be used as an indicator of the maturity of adipocytes. Lipid droplets are intracellular organelles that store lipids such as triglycerides (neutral fats) and cholesterol, and have a droplet-like shape due to the lipids being covered by a single membrane of phospholipids. Furthermore, proteins specific to adipose tissue (such as perilipin) are expressed on the surface of the phospholipids. The size of lipid droplets in mature adipocytes varies, but for example, if the average lipid droplet size is 20 μm or greater, the adipocytes can be considered to be somewhat mature, i.e., mature adipocytes.

[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, "vascular endothelial cells" refer to flat cells that form the surface of the lumen of blood vessels. Examples of vascular endothelial cells include human umbilical vein-derived 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 adipocytes and vascular endothelial cells, but may also include cells other than adipocytes and vascular endothelial cells. Examples of cells other than adipocytes and vascular endothelial cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, cancer cells such as colon cancer cells (e.g., human colon cancer cells (HT29)) and liver cancer cells, cardiomyocytes, epithelial cells (e.g., human gingival epithelial cells), lymphatic endothelial cells, nerve cells, dendritic cells, hepatocytes, adherent cells (e.g., immune cells), smooth muscle cells (e.g., aortic smooth muscle cells (Arota-SMC)), pancreatic islet cells, and keratinocytes (e.g., human epidermal keratinocytes).

[0022] The cell number ratio 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 this embodiment has a vascular network between cells. "Having a vascular network between cells" means having a structure in which branched blood vessels extend between cells, surrounding the cells, similar to biological tissue. Whether a vascular network similar to that of biological tissue is formed can be determined, for example, based on the number of vascular branches and / or the length between vascular branches and / or the variability of vascular diameter in biological tissue. For example, the number of vascular branches in the cell structure may be determined to be similar to that of biological tissue when the average number of vascular branches in the cell structure is 80% to 150%, 85% to 130%, or 90% to 120% of the average number of vascular branches in biological tissue. Furthermore, the number of vascular branches in the cell structure may be determined to be similar to that of biological tissue when the average number of vascular branches in the cell structure is 2.5 to 4.5, or 3.0 to 4.2. For example, when the average length between branching blood vessels in a cell structure is 80% to 150%, 85% to 130%, or 90% to 120% of the average length between branching blood vessels in biological tissue, the cell structure may be determined to be similar to the length between branching blood vessels in biological tissue. Both large and small blood vessels are observed in biological tissue. Therefore, for example, when both large diameter blood vessels (e.g., 10 μm to 25 μm) and small diameter blood vessels (e.g., greater than 0 μm and less than 10 μm) are observed, similar to biological tissue, the cell structure may be determined to have a similar diversity of blood vessel diameters to biological tissue. Furthermore, when 60% or more, 70% or more, or 80% or more of the total blood vessel diameters are distributed in the range greater than 0 μm and less than 25 μm, the cell structure may be determined to have a similar diversity of blood vessel diameters to biological tissue. The cell structure according to this embodiment preferably has a vascular network between adipocytes. In this case, it is preferable that not only the vascular network is present, but also the adipocytes surrounded by the blood vessels are similar to those in biological tissue. For example, if 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 contains 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) As used herein, the term "extracellular matrix component" refers to an assembly of extracellular matrix molecules formed by multiple extracellular matrix molecules. Extracellular matrix refers to a substance present 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. In this embodiment, the extracellular matrix component is preferably a substance present outside animal cells, i.e., an animal extracellular matrix component. The extracellular matrix molecule may be a modified or variant of the above-mentioned extracellular matrix molecule or 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 aggregates of extracellular matrix components into smaller sizes. Fragmented extracellular matrix components may include defibrated extracellular matrix components. Defibrated extracellular matrix components are components obtained by defibrating the above-mentioned extracellular matrix components by applying 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 extracellular matrix components such as collagen components is not particularly limited, and fragmentation may be performed by applying a physical force. The method for fragmenting extracellular matrix components may be, for example, a method for finely breaking down clumped extracellular matrix components. Extracellular matrix components may be fragmented in a solid phase or in an aqueous medium. For example, extracellular matrix components may be fragmented by applying a physical force using an ultrasonic homogenizer, a stirring homogenizer, or a high-pressure homogenizer. When using a stirring homogenizer, the extracellular matrix components may be homogenized directly or in an aqueous medium such as physiological saline. Furthermore, millimeter-sized or nanometer-sized fragmented extracellular matrix components can be obtained by adjusting the homogenization time, number of times, etc.

[0027] The diameter and length of fragmented extracellular matrix components can be determined by analyzing 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, from the viewpoint of facilitating the formation of thick tissue, 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. 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 as a whole is within the above-mentioned range. Specifically, it is preferable that the average lengths of 50% or more of the fragmented extracellular matrix components are within the above-mentioned range, and it is even more preferable that the average lengths of 95% of the fragmented extracellular matrix components are 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 fragmented extracellular matrix components can be determined by measuring individual fragmented extracellular matrix components using an optical microscope or the like and analyzing the images. As used herein, "average length" refers to the average length of the measured sample in the longitudinal direction, and "average diameter" refers to the average length of the measured sample in the direction perpendicular to the longitudinal direction.

[0031] When the extracellular matrix component is a collagen component, the fragmented extracellular matrix component is also referred to as a "fragmented collagen component." The "fragmented collagen component" refers to a collagen component, such as a fibrous collagen component, that has been fragmented and that maintains its 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 crosslinking methods include physical crosslinking by applying heat, ultraviolet light, radiation, etc., and chemical crosslinking using 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 contains a collagen component, crosslinks may be formed between collagen molecules (triple helix structure) or between collagen fibrils formed by the collagen molecules. Crosslinking may be thermal crosslinking (thermal crosslinking). Thermal crosslinking can be performed, for example, by heat treatment under reduced pressure using a vacuum pump. When thermal crosslinking of a collagen component is performed, the extracellular matrix component may be crosslinked by forming a peptide bond (-NH-CO-) between an amino group of the collagen molecule and a carboxy group of the same or another collagen molecule.

[0035] Extracellular matrix components can also be crosslinked using a crosslinking agent. The crosslinking agent may be, for example, one capable of crosslinking carboxyl groups with amino groups, or one capable of crosslinking amino groups with each other. Aldehyde-based, carbodiimide-based, epoxide-based, and imidazole-based crosslinking agents are preferred from the standpoints of economy, safety, and ease of use. Specific examples of crosslinking agents 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 as appropriate depending on the type of extracellular matrix component, the crosslinking method, etc. The degree of crosslinking may be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, or 30% or less, 20% or less, or 15% or less. A crosslinking degree within the above range allows the extracellular matrix molecules to be adequately dispersed, and also provides good redispersibility after dry storage.

[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, etc. 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, the degree of crosslinking measured by the TNBS method is preferably 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 measured 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% by mass, preferably 10 to 90% by mass, preferably 10 to 80% by mass, preferably 10 to 70% by mass, preferably 10 to 60% by mass, preferably 1 to 50% by mass, preferably 10 to 50% by mass, more preferably 10 to 30% by mass, and more preferably 20 to 30% by mass, based on the cell structure (dry weight).

[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" refer 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" refer 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 be derived from the same or different animal species as the endogenous extracellular matrix components. Examples of animal species 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 referred to as an "exogenous collagen component." The "exogenous collagen component," meaning a collagen component supplied from the outside, is an aggregate of collagen molecules formed by a plurality of collagen molecules, and specific examples thereof include fibrous collagen and non-fibrous collagen. The exogenous collagen component is preferably fibrous collagen. The fibrous collagen refers to 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 commercially available, and a specific example thereof is type I collagen derived from porcine skin manufactured by Nippon Meat Packers, Inc. An example of exogenous non-fibrous collagen is type IV collagen.

[0044] The exogenous extracellular matrix components may be derived from a different animal species than the cells. Also, when the cells include extracellular matrix-producing cells, the exogenous extracellular matrix components may be derived from a different animal species than the extracellular matrix-producing cells. In other words, the exogenous extracellular matrix components may be heterologous extracellular matrix components.

[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 endogenous extracellular matrix components and fragmented extracellular matrix components. The content of extracellular matrix components 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 a cell structure is a collagen component, the amount of collagen component in the cell structure can be quantified, for example, by quantifying hydroxyproline as follows. A lysis solution containing the cell structure is mixed with hydrochloric acid (HCl), incubated at high temperature for a predetermined time, then returned to room temperature. After centrifugation, 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 serially diluted to prepare standards. The sample and standard are each treated as required 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. Alternatively, the cell structure may be directly suspended in high-concentration hydrochloric acid, the resulting lysis solution is centrifuged, and the supernatant is recovered and used for quantification of collagen component. The cell structure to be lysed may be in the state it was recovered from the culture medium, or it may be lysed after recovery and drying to remove the liquid components. However, when quantifying collagen components by dissolving cell structures in the state they have been recovered from the culture medium, it is expected that the measured weight of the cell structures will vary due to the influence of medium components absorbed by the cell structures and residual medium due to problems with the experimental technique. Therefore, from the viewpoint of stably measuring the weight of the structure 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 includes, for example, the following method. (Sample preparation) The entire freeze-dried cell structure is mixed with 6 mol / L HCl and incubated in a heat block at 95°C for at least 20 hours, then returned to room temperature. After centrifugation at 13,000 g for 10 minutes, the supernatant of the sample solution is collected. After diluting with 6 mol / L HCl appropriately so that the results fall within the range of the calibration curve in the measurement described below, 200 μL is diluted with 100 μL of ultrapure water to prepare the sample. 35 μL of sample is used.

[0048] (Preparation of standards) Add 125 μL of the standard solution (1200 μg / mL in acetic acid) and 125 μL of 12 mol / L HCl to a screw-cap tube, mix, and incubate at 95°C in a heat block for 20 hours. Then, return the mixture to room temperature. After centrifugation at 13,000×g for 10 minutes, dilute the supernatant with ultrapure water to make S1 (300 μg / mL). Then, serially dilute S1 to make S2 ​​(200 μg / mL), S3 (100 μg / mL), S4 (50 μg / mL), S5 (25 μg / mL), S6 (12.5 μg / mL), and S7 (6.25 μg / mL). Also prepare S8 (0 μg / mL) containing 90 μL of 4 mol / L HCl alone.

[0049] (Assay) Add 35 μL of each standard and sample to a plate (included in the QuickZyme Total Collagen Assay kit, QuickZyme Biosciences). Add 75 μL of assay buffer (included in the kit) to each well. Seal the plate and incubate at room temperature for 20 minutes with shaking. Remove the seal and add 75 μL of detection reagent (reagent A:B = 30 μL:45 μL, included in the kit) to each well. Seal the plate, mix the 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 component is calculated by comparing the sample absorbance 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 constituents using a known staining method (e.g., immunostaining using an anti-collagen antibody or Masson's trichrome staining), and then calculating the ratio of the area where the collagen component is present in the entire cell structure using macroscopic observation, various microscopes, image analysis software, etc. When defining by area ratio, there are no limitations on which cross section or surface in the cell structure is used to define the area ratio. For example, if the cell structure is a sphere, it may be defined by a cross section passing through the approximate center of the cell structure.

[0051] For example, when the collagen component in a cell structure is defined by its area ratio, the area ratio is 0.01 to 99% of 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 term "collagen component in a cell structure" is as described above. The area ratio of the collagen component constituting the cell structure refers to 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 blue-stained collagen component to the total area of ​​a cross section passing through approximately the center of the cell structure.

[0052] The cell structures preferably have 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 stable and resistant to enzymatic degradation during or after culture. The survival rate can be calculated, for example, from the mass of the cell structures 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, pH 7.4, and for a reaction time of 15 minutes. Such cell structures are stable and resistant to enzymatic degradation during or after culture.

[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, but 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 irregularities, the thickness means the distance at the thinnest part of the main surface.

[0056] Furthermore, when the cell structure is spherical or approximately spherical, it means its diameter. Furthermore, when the cell structure is ellipsoidal or approximately ellipsoidal, it 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 the 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 produced when thrombin acts on fibrinogen, releasing the A and B chains from the N-termini of the Aα and Bβ chains. Fibrin is a polymer that 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 among cells according to this 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 bringing fragmented extracellular matrix components into contact 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. In (i), cells other than adipocytes, stem cells, and vascular endothelial cells may be included, and in (ii), cells other than adipose stem cells and vascular endothelial cells may be included. The stem cells in (i) are preferably adipose stem cells. Furthermore, the adipocytes preferably include mature adipocytes.

[0061] By dispersing the fragmented extracellular matrix components in an aqueous medium, they can be more easily brought into contact with cells in the aqueous medium, thereby promoting 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 contacting steps 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 pre-prepared aqueous medium.

[0063] 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, stem cells and vascular endothelial cells may be added to the aqueous medium containing the fragmented extracellular matrix components, followed by the addition of adipocytes; stem cells, vascular endothelial cells, and adipocytes may be added to the aqueous medium containing the fragmented extracellular matrix components in that order; 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 addition, mixing may be performed by stirring, or the like, or mixing may not be required. Furthermore, the contacting step may include a step of incubating the cells for a certain period of time after contacting them with the fragmented extracellular matrix components.

[0064] The contacting step may be performed after forming a cell layer in an aqueous medium. That is, the contacting step may be performed by forming a cell layer in an aqueous medium and then contacting the cell layer with an extracellular matrix component. By forming a cell layer before contacting the cell layer with an extracellular matrix component, a cell structure with 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 contacting 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 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) for heating the extracellular matrix components can be determined appropriately. The heating temperature may be, for example, 100°C or higher, 200°C or lower, or 220°C or lower. Specific examples of the heating temperature include 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, and 220°C. The heating time (the time for maintaining the temperature) can be determined appropriately depending on the heating temperature. For example, when heating at 100°C to 200°C, the heating time may be 6 hours or longer and 72 hours or shorter, more preferably 24 hours or longer and 48 hours or shorter. In the crosslinking step, heating may be performed in the absence of a solvent or under reduced pressure.

[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 solution containing the fragmented extracellular matrix components and the aqueous medium. Removal of the aqueous medium does not mean that no water is attached to the fragmented extracellular matrix components, but rather means that water is removed to a degree that can reasonably be 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 of the total number of cells in the contacting step, and may be 95% or less, 90% or less, 80% or less, or 75% or less.

[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 contacting 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 contacting step may be 0.1 to 90% by mass, or 1 to 30% by mass.

[0074] The amount of fragmented extracellular matrix components in the contact step is, for example, 1.0 × 10 6 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 relative to the number of cells, or 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, or 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 contacting step, the mass ratio of extracellular matrix components to cells (extracellular matrix components / cells) is preferably 1 / 1 to 1000 / 1, more preferably 9 / 1 to 900 / 1, and even more preferably 10 / 1 to 500 / 1.

[0076] The cell number ratio 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 during the contacting step or after the contacting step and before the culturing step. When both fibrinogen and thrombin are added, for example, fibrinogen and thrombin may be added simultaneously, or fibrinogen may be added first followed by thrombin. The timing of adding fibrinogen and / or thrombin is not particularly limited. For example, fibrinogen and / or thrombin may be added to an aqueous medium containing stem cells, vascular endothelial cells, adipocytes, and extracellular matrix components, or to an aqueous medium containing stem cells, vascular endothelial cells, and extracellular matrix components. Alternatively, for example, fibrinogen may be added to an aqueous medium containing stem cells, vascular endothelial cells, and extracellular matrix components, followed by adipocytes, and then thrombin. Adding fibrinogen and / or thrombin can suppress shrinkage that may occur during the culturing step described below, making it easier to control the shape and size of the cell structure. Furthermore, the suspension of cells and extracellular matrix components can be gelled, which facilitates detachment of the suspension from a culture vessel (support) after it has been dropped onto the vessel. 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 an uneven state relative to other cells. However, by gelling the suspension, it becomes easier to maintain a uniform mixture of each cell and extracellular matrix component, and to maintain the cells and extracellular matrix components in close proximity to each other.

[0078] The method may further include a step of sedimenting the extracellular matrix components and cells together 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 cells in the cell structure becomes more uniform. Specific methods for this include, but are not limited to, centrifuging the culture solution containing the extracellular matrix components and 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 cells in contact with 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 cells in contact with fragmented extracellular matrix is ​​not particularly limited and may be, for example, a well insert, a low-adhesion plate, or a plate with a U- or V-shaped bottom. 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 after being detached from the support during the culture, it is preferable to use a plate with a U- or V-shaped bottom that inhibits cell adhesion to the support, or a low-adhesion plate.

[0082] The medium is not particularly limited, and an appropriate medium can be selected depending on the type of cells to be cultured. Examples of media include Eagle's MEM medium, DMEM, Modified Eagle Medium (MEM), Minimum Essential Medium, RPMI, and GlutaMax medium. The medium may be a serum-supplemented medium or a serum-free medium. The medium may also be a mixed medium made by mixing two types of media.

[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, and 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 of 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, using the following formula: In the formula, L1 represents the length of the longest part of the cell structure on day 1 of culture, and L3 represents the length of the corresponding part of the cell structure on day 3 of culture. Shrinkage rate (%) = {(L1-L3) / L1} x 100

[0085] In the above example, the contraction rate was calculated from the cell structure on day 1 and day 3 of culture, but it may also be calculated from the cell structure at any time during the culture period, including the end of culture. For example, it may be calculated from the cell structure on day 1 and day 2 of culture, or from the cell structure on day 1 and day 5 of culture, or from the cell structure on day 1 and day 8 of 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 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 second culture step may be the same type as the cells used in the first contact step and first culture step, or may be a different type. A two-layered cell structure can be produced by the second contact step and second culture step. Furthermore, by repeatedly including the contact step and culture step, a multi-layered cell structure can be produced, and tissues that are more similar to those of a more complex living organism can also be produced.

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

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

[0089] The method for detaching cells in contact with the fragmented extracellular matrix from the support is not particularly limited, and may involve, for example, using a low-adhesion support and detaching the cells from the support by adding a culture medium, directly physically detaching the cells from the support using an instrument or the like, detaching the cells from the support by applying vibration, or using a support 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 detaching the cells from the support by applying the stimuli. When detaching cells from the support by adding a culture medium, the culture medium exemplified above can be used.

[0090] In the culture process, methods for culturing the cells in contact with the fragmented extracellular matrix without them being attached to a support from the beginning include, for example, a method in which a suspension containing 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 highly viscous solvent, and then the solvent alone is removed and the cells are transferred to a culture vessel.

[0091] In the above-mentioned culture process, the timing of 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] [Cellular tissue and its manufacturing method] By using a plurality of the above-mentioned cell structures that are aggregated in a mass without being attached to a support, i.e., "cell structures that contain fragmented extracellular matrix components and cells, have a vascular network between the cells, and are aggregated in a mass without being attached to a support, wherein the cells include at least fat cells and vascular endothelial cells," it is possible to produce cellular tissue in which the vascular network connects the plurality of cell structures.

[0094] The production of the cell tissue involves suspension culturing the cell structures not attached to multiple supports. The multiple cell structures adhere to each other during the suspension culture process, and vascular networks are connected between the cell structures, allowing for the easy production of large cell tissues with connected vascular networks. The number and size of the cell structures used can be appropriately selected depending on the intended use of the cell tissue, the desired size of the cell tissue, and other factors. The type of medium (culture solution) and culture conditions used for suspension culture can also be appropriately selected, and the culture can be carried out using, for example, the medium and conditions exemplified above (culture step).

[0095] [Uses of cell structures] As described above, the cell structure according to this embodiment has a vascular network formed between cells, similar to that of living tissue, and is expected to be easily engrafted when transplanted into animals such as mammals, making it suitable for transplantation. The cell structure used for transplantation may be one or more. When multiple cell structures are used, for example, 1 to 1,000, 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, such as a human, or a non-human animal such as a monkey, dog, cat, rabbit, pig, cow, mouse, or rat.

[0097] The transplantation method according to this embodiment includes transplanting the cell structure having the vascular structure according to this embodiment into an animal. The transplantation method may further include preparing the animal to receive the transplant and / or preparing the cell structure by the above-described method before the transplantation. The transplantation method is not particularly limited and can be performed using a known surgical method appropriate to the recipient, etc. Examples of surgical methods include incising the skin of the recipient and directly transplanting the cell structure under the skin, or injecting the cell structure under the skin of the recipient using a syringe, etc. The cell structure to be transplanted may be one or more, or may be a cellular tissue containing multiple cell structures. The cell structure or cellular tissue may be collected from a culture medium. Depending on the type of transplantation, the cell structure or cellular tissue may be, for example, gelled or semi-gelled (e.g., fibrin gel), or a dispersion in which multiple cell structures are dispersed may be used. Alternatively, fibrin may be added to a cell structure collected from a culture medium and aggregated into a mass without adhering to multiple supports, and the resulting 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 recipient'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 recipient 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 recipient's own blood vessels, resulting in 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. The method may further include preparing a non-human animal to be transplanted and / or producing a cell structure by the above-described method before the transplantation. The transplantation method is as described above. The cell structure to be transplanted may be one or more, or may be a cellular tissue containing multiple cell structures. The method for producing a non-human model animal according to the present embodiment may include, after transplanting the cell structure into the non-human animal, growing the non-human animal for, for example, 7 days or more, 30 days or more, or 90 days or more. The non-human model animal according to the present embodiment can be used to apply data obtained from animal experiments to humans. To produce a non-human model animal, it is preferable to use a non-human animal in which rejection of the transplant (graft) has been suppressed, for example, a non-human animal with weakened or immunodeficient immune system. The non-human model animal can be used, for example, as a pathological in vitro model for adipose tissue-related inflammatory diseases, or for screening pharmaceuticals for diabetes, obesity, etc., and for assay screening of cosmetics for cellulite, obesity, etc.

[0100] The cell structure according to this embodiment can itself be used as a substitute for experimental animals, transplant material, etc. Specific examples include tissue reconstruction, pathological in vitro models, pharmaceutical screening (drug evaluation), cosmetic assay screening, etc., as described above.

[0101] [Methods for evaluating drug effects and screening methods] As described above, the cell structure according to this embodiment has a structure in which branched blood vessels extend between cells so as to surround the cells, similar to biological tissue. Therefore, the cell structure according to this embodiment can be used to evaluate the effects of drugs that inhibit or promote the metabolism of adipose tissue, and to screen drugs. One embodiment of the present invention provides a method for evaluating the effect of a drug that inhibits or promotes adipose tissue metabolism using a cell structure, the method comprising: an administration step of administering a drug that inhibits or promotes adipose tissue metabolism to a cell structure; and an evaluation step of evaluating the effect of the drug based on metabolic changes in the cell structure to which the drug has been administered. According to this embodiment, the effect of a drug that inhibits or promotes adipose tissue metabolism can be effectively evaluated.

[0102] In the administration step, a drug that inhibits or promotes adipose tissue metabolism is administered to the cell structure. The drug may be a drug known to inhibit or promote adipose tissue metabolism, or may be a drug not known to inhibit or promote adipose tissue metabolism.

[0103] Examples of drugs that inhibit adipose tissue metabolism include insulin and TNFα inhibitors. Insulin is known to promote the uptake of fatty acids and glucose. Furthermore, adipocytes mainly take up fatty acids in vivo via FATP-1 and FATP-4 transporters, and TNFα is known to inhibit the function of these transporters and suppress fatty acid uptake. Examples of drugs that promote adipose tissue metabolism include apigenin, cytochalasin B, and isoproterenol. In vivo, adipocytes mainly take up glucose via GLUT-1 and GLUT-4 transporters, and apigenin and cytochalasin B are known to inhibit the function of these transporters and suppress glucose uptake. Furthermore, catecholamines promote the release of taken-up fatty acids and glucose, and isoproterenol, an artificially synthesized catecholamine, is known to function as a fatty acid and glucose release promoter. Drugs that inhibit or promote adipose tissue metabolism may be fatty acid and / or glucose uptake promoters or inhibitors, or fatty acid and / or glucose release promoters or inhibitors, other than those mentioned above.

[0104] The administration of a drug that inhibits or promotes the metabolism of adipose tissue may be carried out by using a medium containing the drug as the medium for culturing the cell structure, or by adding the drug to the medium for culturing the cell structure.

[0105] The cell structure to which a drug that inhibits or promotes adipose tissue metabolism is administered may be a cell structure that has been cultured for one day or more, may be a cell structure that has been cultured for five days or more, may be a cell structure that has been cultured for six days or more, may be a cell structure that has been cultured for seven days or more, may be a cell structure that has been cultured for a longer period of time, or may be a cell structure that has been cultured for a period of 7 days or more but not more than 14 days.

[0106] In the evaluation step, the efficacy of a drug is evaluated based on changes in metabolism in the cell structure to which the drug is administered. The efficacy of a drug can be evaluated using as an index changes in metabolism in the cell structure, for example, changes in the uptake of glucose and / or fatty acids and / or changes in the release of the taken-up glucose and / or fatty acids. By using changes in metabolites other than glucose and fatty acids in adipose tissue as an index, other metabolic systems in adipose tissue can also be evaluated. The metabolic change in the cell structure may be, for example, a change in the amount of glucose and / or fatty acids taken up per unit time in the cell structure and / or a change in the amount of glucose and / or fatty acids released per unit time in the cell structure. The efficacy of a drug may be evaluated based on changes in only one of the above indexes, or may be evaluated based on changes in two or more of the above indexes. The metabolic change may be evaluated by qualitative or quantitative comparison.

[0107] The evaluation step can be performed, for example, by comparing the metabolism in a cell structure to which the drug has been administered with the metabolism in a cell structure to which the drug has not been administered.

[0108] The evaluation step may be carried out multiple times, i.e., the evaluation of the efficacy may be carried out multiple times at predetermined intervals after administration of the drug.

[0109] In the evaluation step, for example, if the metabolism in the cell structure administered with the drug is lower compared to the metabolism in the cell structure not administered with the drug, the drug may be evaluated as being effective as a drug that inhibits adipose tissue metabolism, whereas if the metabolism in the cell structure administered with the drug is higher or unchanged, the drug may be evaluated as being ineffective as a drug that inhibits adipose tissue metabolism.Furthermore, for example, if the metabolism in the cell structure administered with the drug is higher compared to the metabolism in the cell structure not administered with the drug, the drug may be evaluated as being effective as a drug that promotes adipose tissue metabolism, whereas if the metabolism in the cell structure administered with the drug is lower or unchanged, the drug may be evaluated as being ineffective as a drug that promotes adipose tissue metabolism.

[0110] Furthermore, one embodiment of the present invention provides a method for screening for drugs that inhibit or promote adipose tissue metabolism using a cell structure. According to this embodiment, drugs that inhibit or promote adipose tissue metabolism can be effectively selected.

[0111] A screening method for drugs that inhibit or promote adipose tissue metabolism using cell structures may include a step of selecting a drug that is evaluated to be effective as a drug that inhibits or promotes adipose tissue metabolism in the evaluation step of the above evaluation method. The screening method may also include, for example, a step of measuring metabolism in a cell structure to which a drug has been administered; A step of comparing the metabolism in the cell structure to which the drug has been administered with the metabolism in the cell structure to which the drug has not been administered, and selecting the drug as a candidate drug for inhibiting adipose tissue metabolism if the metabolism in the cell structure to which the drug has been administered is lower, or Measuring metabolism in the cell structure to which the drug has been administered; The method can include a step of comparing the metabolism in the cell structure to which the drug has been administered with the metabolism in the cell structure to which the drug has not been administered, and if the metabolism in the cell structure to which the drug has been administered is higher, selecting the drug as a candidate drug that promotes the metabolism of adipose tissue.

[0112] Comparison of metabolism in cell structures can be performed using, for example, the uptake of glucose and / or fatty acids and / or the release of the taken-up glucose and / or fatty acids as indicators. Changes in metabolites other than glucose and fatty acids in adipose tissue can also be used as indicators. Comparison of metabolism in cell structures can be performed using, for example, the amount of glucose and / or fatty acids taken up per unit time in the cell structure and / or the amount of glucose and / or fatty acids released per unit time in the cell structure as indicators. Metabolism in cell structures can be compared based on only one of the above indicators, or based on two or more of the above indicators. Comparison can be qualitative or quantitative.

[0113] The candidate substance may be, for example, a fatty acid and / or glucose uptake promoter or inhibitor, a fatty acid and / or glucose release promoter or inhibitor, or an uptake promoter or uptake inhibitor, or release promoter or release inhibitor of a metabolite other than glucose and fatty acids.

[0114] The comparison of the metabolism in the cell structure may be performed multiple times, i.e., the comparison of the metabolism in the cell structure may be performed multiple times at predetermined intervals after administration of the drug.

[0115] The administration of drugs that inhibit or promote adipose tissue metabolism can be carried out in the same manner as in the evaluation method described above. The cell structures and the like used can also be those described above. [Example]

[0116] <Test Example 1: Preparation of defibrated collagen component> A collagen component in which at least a portion was cross-linked (cross-linked collagen component) was obtained by heating 100 mg of porcine skin-derived collagen type I sponge fragments (manufactured by Nippon Meat Packers, Inc.) at 200°C for 24 hours. No significant changes in the appearance of the collagen were 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 mixture was homogenized for 6 minutes using a homogenizer (VH-10, AS ONE Corporation) to defibrate the cross-linked collagen component.

[0117] The collagen solution 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 sonicated at 100 V for 20 seconds using a sonicator (Sonics and Materials, VC50) while keeping it on ice. After removing the sonicator, the tube containing the collagen solution was cooled on ice for 10 seconds, and this cycle was repeated 100 times. After 100 cycles of sonication, the collagen solution was filtered through a 40 μm pore size filter to obtain a dispersion containing defibrated collagen component (sCMF). The dispersion was lyophilized by conventional methods to obtain the defibrated collagen component (sCMF) as a dry product. The average length of the sCMF was 14.8 ± 8.2 μm (N = 20).

[0118] <Test Example 2: Preparation and evaluation of cell structures (1)> The cells, reagents, and preparation method used in the preparation of the cell structures are as follows. (cells and collagen) Human adipose tissue (derived from thigh) for obtaining primary human mature adipocytes and human adipose stem cells (ADSCs) (provided by Kyoto Prefectural University of Medicine Hospital) Human umbilical vein-derived endothelial cells (HUVEC) (Lonza #C-2517A) Defibrillated collagen component (sCMF) (prepared in Test 1)

[0119] (reagent) Bovine pancreatic insulin (Sigma #I1882) Bovine plasma-derived thrombin lyophilized powder (Sigma #T4648) Clostridium histolyticum collagenase type I (Sigma #C0130) Bovine plasma-derived fibrinogen type IS (Sigma #F8630) DMEM (high glucose, Nacalai Tesque) EGM-2MV Bullet Kit with growth factors (Lonza #C-2517A)

[0120] (Culture medium and various solutions) EGM-2 medium: 500 mL of EGM-2 mixed with EGM-2 supplement growth factors and stored at 4°C 10 mg / mL insulin stock solution: 100 mg of the above bovine pancreatic insulin was dissolved in 10 mL of 1% glacial acetic acid solution (pH ≦ 2) diluted with water, and the solution was dispensed into Eppendorf tubes in equal amounts and stored at -20°C. 2 mg / mL collagenase solution: Mix 2.5 g of BSA with 50 mL of DMEM (0% FBS, 1% antibiotics). To digest the adipose tissue in a 6-well plate, mix 26 mg of collagenase type I with 13 mL of DMEM (0% FBS, 5% BSA, 1% antibiotics) and filter through a 0.2 μm 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, place in a 37°C water bath for 3-5 minutes, filter through a 0.2µm filter, and dispense equal volumes into Eppendorf tubes for use. 202U / mL thrombin stock solution: Weigh 202U of thrombin into an Eppendorf tube, immediately add 1mL of DMEM (0% FBS, 1% antibiotics), and dissolve in a 37°C water bath for 3-5 minutes. Then, filter through a 0.2μm pore size filter and dispense equal amounts into Eppendorf tubes for use.

[0121] (Production method) Human adipose tissue fragments were washed with PBS containing 5% antibiotics. 4–6 g of tissue was divided into six wells of a 6-well plate. Using scissors and tweezers, the tissue was minced into approximately 1–3 mm pieces in 2 mL of 2 mg / mL collagenase solution. After 1 hour of incubation at 37°C and 230 rpm, the tissue was mixed for 30 minutes with a 10 mL pipette. The lysate was filtered through a 500 μm pore-size iron mesh filter, and 2 mL of DMEM was added per well to recover all digested cells. The lysate was then centrifuged at 200 g for 3 minutes 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. 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). The cells were washed twice with 25 mL of DMEM.

[0122] The upper layer containing mature adipocytes was collected and placed in an Eppendorf tube. Nuclei were stained with Hoechst stain (1:1000 diluted Hoechst, stained for 15 minutes), and the cell number was counted using a Turker Burk hemocytometer under a fluorescent microscope.

[0123] The pellet containing ADSCs was suspended in 10 mL of DMEM and seeded into a 10 cm dish for subculture. The ADSCs were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and counted.

[0124] HUVECs purchased from Lonza were suspended in 10 mL of DMEM, seeded in a 10 cm dish, and subcultured. HUVECs were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and the cell number was counted.

[0125] One mg of sCMF was weighed, 100 μL of DMEM was added, and gently mixed until only small particles of sCMF were observed. The mixture was centrifuged at 10,000 rpm at room temperature for 1 minute, and the supernatant was aspirated to obtain an sCMF pellet. 250,000 ADSCs and 125,000 HUVECs (ADSC:HUVEC = 2:1) were gently added to 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 a 50 mg / mL fibrinogen stock solution) was added and gently mixed with the cells and sCMF. 300,000 mature adipocytes were then added and gently mixed. If necessary, a small amount of DMEM was added to adjust the total volume to 70 μL. Immediately, 0.15 U of thrombin (0.71 μL of a 202 U / mL thrombin stock solution) was added, mixed, and then the mixture was slowly seeded into a transwell placed on a 6-well adapter on a 6-well plate.

[0126] The cells were incubated in a 37°C incubator for 1 hour to allow gelation, and 12 mL of EGM-2 medium containing insulin at a final concentration of 10 μg / mL was added. The medium was replaced every 2–3 days until day 7 of culture.

[0127] 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 (500 μL inside the transwell, 500 μL outside the transwell) at room temperature for 7 minutes and then washed three times with 2 mL of PBS.

[0128] The tissues were blocked with 1% BSA / PBS solution at room temperature for 1 hour (500 μL into the transwell, 500 μL outside the transwell). The BSA solution was aspirated, and 100 μL of primary antibody solution (CD31 and perilipin diluted 1:100 in 1% BSA / PBS) was added (50 μL into 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 secondary antibody solution (AlexaFluor 647-labeled anti-mouse anti-CD31 antibody and AlexaFluor 488-labeled anti-rabbit anti-perilipin antibody diluted 2:200 in 1% BSA / PBS, Hoechst diluted 1:1000) was added (50 μL into 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, followed by washing four times with 2 mL of PBS.

[0129] 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 at 640 nm (AlexaFluor 647) and 488 nm (AlexaFluor 488).

[0130] The diameter of lipid droplets in the constructed 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.

[0131] Figure 1 shows the results of fluorescence imaging of a cell structure containing a vascular network (20x magnification). (a) shows adipose tissue extracted from a living organism and fixed as is, and (b) shows the cell structure. In the cell structure, a vascular network was observed surrounding mature adipocytes (large, round, single-locular lipid droplets), just like in living tissue. These vascular networks extended deep into the cell structure. The average diameter of the lipid droplets was 85 μm (N = 50), which was similar to the average diameter of mature adipocytes in living tissue (72 μm (N = 50)). Furthermore, the formed blood vessels were hollow, just like in living tissue, and both large diameters (e.g., 10 μm to less than 25 μm) and small diameters (e.g., greater than 0 μm and less than 10 μm) were observed, just like in living tissue. As shown in Figure 2, the number of vascular branches was also found to be similar to that in living tissue. Furthermore, the distribution of the length between blood vessel branches was 0-100 μm: 42.5%, 100-200 μm: 39.4%, and 200 μm or more: 18.1% (N=180), which was found to be similar to the distribution of the length between blood vessel branches in the vascular network in biological tissues (0-100 μm: 61.8%, 100-200 μm: 24.7%, and 200 μm or more: 13.5% (N=180)). In Test Example 2, the length between blood vessel branches was frequently between 50 μm and 100 μm, 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 adipocytes in adipose tissue in vivo, and blood vessels are formed between the adipocytes. The distribution of the lengths between the branching blood vessels as described above suggests that the cell structure of Test Example 2 may be able to faithfully mimic actual in vivo adipose tissue.

[0132] <Test Example 3: Preparation and evaluation of cell structures (2)> A cell structure was prepared 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. A cell structure was prepared in which a vascular network was formed surrounding the mature adipocytes. Figure 3 shows the results of fluorescence 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 prepared even when the number of mature adipocytes and the amount of sCMF were changed.

[0133] <Test Example 4: Preparation and evaluation of cell structures (3)> A cell structure was prepared using the same method as in Test Example 2, except that mature adipocytes were not used and 2 mg of sCMF, 0.6 mg of fibrinogen, and 0.3 U of thrombin were used. A cell structure with a vascular network was successfully prepared. Figure 4 shows the results of fluorescent observation (4x magnification) of a cell structure in which only the blood vessels were stained using an anti-CD31 antibody. This demonstrates that a cell structure with a vascular network can be prepared without using mature adipocytes.

[0134] <Test Example 5: Preparation and evaluation of cell structures (4)> A cell structure was prepared using the same method as in Test Example 2, except that mature adipocytes were not used and 50,000 HUVEC cells (ADSC:HUVEC = 5:1), 2 mg of sCMF, 0.6 mg of fibrinogen, and 0.3 U of thrombin were used. A cell structure with a vascular network was successfully prepared. Figure 5 shows the results of fluorescence observation of a cell structure in which only the blood vessels were stained using an anti-CD31 antibody (4x magnification). This demonstrates that a cell structure with a vascular network can be prepared even when mature adipocytes are not used and the ratio of ADSC to HUVEC is changed.

[0135] <Test Example 6: Preparation and evaluation of cell structures (5)> Cell constructs were prepared using the same method as in Test Example 2, except that adipose tissue obtained by liposuction from human thighs (biological tissue) was used instead of mature adipocytes, ADSCs, and HUVECs, and the total volume before seeding was adjusted to 60 μL. For the adipose tissue, 3 g of collected biological tissue was minced into pieces approximately 1–3 mm in size using scissors and tweezers, and then liquefied by slowly pipetting several times using a 10 mL syringe. 60 μL of the liquefied adipose tissue was then mixed with sCMF. The adipose tissue obtained by liposuction contained mature adipocytes, adipose stem cells, and vascular endothelial cells. Figure 6 shows the results of fluorescence observation (10x magnification) of cell constructs stained using the same method as in Test Example 2. It was demonstrated that cell constructs with vascular networks could be prepared using adipose tissue obtained from biological tissue instead of mature adipocytes, ADSCs, and HUVECs. It was also confirmed that cell constructs with vascular networks could be prepared using 2 mg of sCMF.

[0136] In Test Examples 2 to 6, it was confirmed that cell structures with vascular networks could be produced in all cases, but a comparison of Test Examples 2 to 6 showed that using 1 mg of sCMF tended to produce cell structures with vascular networks more similar to living tissue than using 2 mg of sCMF. Furthermore, when the ratio of ADSC to HUVEC was ADSC:HUVEC=2:1, cell structures with vascular networks more similar to living tissue tended to be produced than when ADSC:HUVEC=5:1.

[0137] <Comparative Example> A cell construct was prepared by the same method as in Test Example 2, except that sCMF was not used, and 1 mg of fibrinogen and 0.5 U of thrombin were used. No blood vessel formation was observed in the prepared cell construct. Furthermore, a cell construct was prepared by the same method as in Test Example 2, except that no ADSCs were included. Only very slight blood vessel formation was observed in the prepared cell construct, and the formation of a vascular network surrounding mature adipocytes could not be confirmed.

[0138] <Test Example 7: Preparation and evaluation of cell structures (6)> The 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 using the same method as in Test Example 1. An overview of this test example is shown in Figure 7. In Figure 7, the circles in the droplet on the left represent mature adipocytes, the white diamonds represent ADSCs, and the gray short bars represent HUVECs.

[0139] (Production method) Human adipose tissue fragments were washed with PBS containing 5% antibiotics. 4–6 g of tissue was divided into six wells of a 6-well plate. Using scissors and tweezers, the tissue was minced into approximately 1–3 mm pieces in 2 mL of 2 mg / mL collagenase solution. After 1 hour of incubation at 37°C and 230 rpm, the tissue was mixed for 30 minutes with a 10 mL pipette. The lysate was filtered through a 500 μm pore-size iron mesh filter, and 2 mL of DMEM was added per well to recover all digested cells. The lysate was then centrifuged at 200 g for 3 minutes 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. 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). The cells were washed twice with 25 mL of PBS (5% BSA, 1% antibiotics) and then with 25 mL of DMEM.

[0140] The upper layer containing mature adipocytes was collected and placed in an Eppendorf tube. Nuclei were stained with Hoechst stain (1:1000 diluted Hoechst, stained for 10 minutes), and the cell number was counted using a Turker Burk hemocytometer under a fluorescent microscope.

[0141] The pellet containing ADSCs was suspended in 10 mL of DMEM and seeded into a 10 cm dish for subculture. The ADSCs were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and counted.

[0142] HUVECs purchased from Lonza were suspended in 10 mL of DMEM, seeded in a 10 cm dish, and subcultured. HUVECs were detached from the dish using trypsin / EDTA, suspended in 1 mL of DMEM, and the cell number was counted.

[0143] To obtain one approximately 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.

[0144] 2.4 mg of sCMF was weighed out, 1 mL of DMEM was added, and gently mixed until only small particles of sCMF were observed. The mixture was centrifuged at 10,000 rpm at room temperature for 1 minute, and the supernatant was aspirated to obtain an sCMF pellet. 220,000 ADSCs and 275,000 HUVECs were gently added to the sCMF pellet, and the mixture was centrifuged at 3,500 rpm at room temperature for 1 minute, and the supernatant was aspirated. Fibrinogen (32 μL of a 50 mg / mL fibrinogen stock solution) was added and gently mixed with the cells and sCMF. 650,000 mature adipocytes were then 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 after brief mixing, the mixture (enough for 40 cell balls) was seeded onto a low-adhesion 96-well plate (IWAKI #4860-800LP) at 5 μL per well (enough for one cell ball).

[0145] The mixture was incubated in an incubator at 37°C 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.

[0146] 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-mentioned EGM-2 medium. The medium was changed every 2–3 days until the 7th day of culture.

[0147] Fluorescence imaging of 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 then 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.

[0148] 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 1:100 in 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 2:200 in 1% BSA / PBS solution, Hoechst diluted 1:1000) was added. The plate was then incubated with a wet tissue placed under the plate, covered with aluminum foil, and incubated at room temperature for 2 hours, followed by four washes with 200 μL of PBS.

[0149] 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 at 640 nm (AlexaFluor 647) and 488 nm (AlexaFluor 488).

[0150] 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 as in Test Example 2, and Nile Red staining was performed by standard methods. Photograph (b) is a further enlargement of one of the cell balls in (a). It was confirmed that vascular networks had formed in the cell balls, surrounding mature adipocytes (large, round, single-locular lipid droplets), just like in living tissue. These vascular networks were even formed deep inside the cell balls.

[0151] Figure 9 shows the results of CD31 immunohistochemical staining of a section of a cell ball with a vascular network. From these results, many lumens were observed in the cell ball, confirming that the vascular network had formed deep into the cell structure.

[0152] Figure 10 shows the average diameter (n=12 cell balls / volume) of cell balls prepared by the method of this test example after 7 days of culture. The average diameter of the 5 μL cell balls was 1256 μm, and the average diameter of the 10 μL cell balls was 1857 μm.

[0153] <Test Example 8: Preparation and evaluation of cellular 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 Figure 11, multiple cell balls (five in Figure 11) aggregated and united to form 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 (Figures 11(a) and (b)).

[0154] <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 three mice for 90 days. (1) A mixture of 111 cultured cell balls 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).

[0155] After 30 days of growth, tissue was collected from the transplantation site of each individual. Adipose tissue was formed in the area where (2) above was transplanted, but no vascular network was observed. Adipose tissue was formed in the area where (3) above was transplanted, and a slight vascular network was observed on the tissue surface, but the presence of large oil droplets was also observed. The formation of oil droplets indicates the death of adipocytes. On the other hand, in the area where (1) above, which used the cell balls 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 droplet formation was observed in the tissue where (1) above was transplanted, indicating better adhesion after transplantation.

[0156] After 30 days of growth, tissue was collected from the transplantation site (1) of the individual and stained for perilipin and DAPI in the same manner as in Test Example 2 (Figure 12). DAPI staining was performed using standard methods. A: SFT indicates tissue collected from the transplantation site (3) above, and C: 3DVFT indicates tissue collected from the transplantation site (1) above. The top row of Figure 12 shows the results of bright field observation, the middle row shows the results of perilipin staining, and the bottom row shows the results of DAPI staining. It was confirmed that mature adipocytes were formed in the tissue formed at the transplantation site.

[0157] In addition, tissue was collected from the transplantation site (1) of the individual after 90 days of growth and stained for perilipin and CD31 in the same manner as in Test Example 2 (Figure 13). The upper row of Figure 13 shows the results of bright field observation, 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 had been formed.

[0158] From the above, it was demonstrated that the cell structure according to this embodiment has excellent adhesion after transplantation and is suitable for transplantation.

[0159] Test Example 10: Preparation of cell constructs and evaluation of glucose uptake (Cell structure creation) A cell structure with a vascular network was prepared using the same procedure as in Experimental Example 2, except that 6,500 mature adipocytes, 5,500 adipose stem cells, 2,750 vascular endothelial cells, 0.025 mg of defibrated collagen component (sCMF), 0.015 mg of fibrinogen, and 0.007 U of thrombin were used per tissue.

[0160] The above materials were prepared in bulk for 96 wells to obtain gels, which were then seeded into each well of a 96-well plate. Two types of sCMF were used: one made using only pig skin-derived collagen type I sponge fragments (manufactured by Nippon Meat Packers, Inc.), and one made using a mixture of pig skin-derived collagen type I sponge fragments and pig skin-derived collagen type III sponge fragments (manufactured by Nippon Meat Packers, Inc.). The mixing ratio of type I collagen to type III collagen in the mixture was estimated to be approximately 8:2. In this test example, the sCMF used was made using a mixture of pig skin-derived collagen type I sponge fragments and pig skin-derived collagen type III sponge fragments.

[0161] The seeded cell structures were cultured until the day of the uptake and release test. The medium used was glucose- and fatty acid-free DMEM medium, and the medium was changed every two days.

[0162] (Assessment of glucose uptake) Tests to evaluate glucose uptake were performed on days 7 and 14 after the formation of the cell constructs.

[0163] Before the experiment, the medium was replaced with 300 μL of glucose-free DMEM medium. The cell constructs were incubated for 6 hours, thereby starving the cell constructs. The medium was replaced with 100 μL of DMEM medium containing 0.1 mg / mL NBD-modified glucose (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, Cayman Chemical). The cell constructs were incubated and fluorescence measurements were performed at 10, 30, 60, 120, and 150 minutes. Before each measurement, the medium in the cell constructs to be measured was replaced with 300 μL of PBS buffer. Fluorescence was measured using a plate reader (Synergy HTX, BIO Tek) and the integrated fluorescence intensity in the image was calculated.

[0164] As shown in Figure 14, it was confirmed that the longer the cell construct was exposed to glucose, the more glucose was taken up into the construct. Furthermore, a comparison of the fluorescence intensity after 60 minutes for cell constructs on the 7th and 14th days of formation is shown in the graph in Figure 15(b). The number of samples for each treatment was 4 (n = 4), and the glucose uptake in the control medium containing no NBD-modified glucose at 0 hours of treatment was set to 100%. These results demonstrate that the glucose uptake ability of the cell construct is maintained for 14 days or more. The graph in Figure 15(a) shows the results using a tissue constructed in the same way as the cell construct in this test example, except that it did not contain vascular endothelial cells, and it was similarly demonstrated that the glucose uptake ability was maintained for 14 days or more.

[0165] Test Example 11: Evaluation of the effects of glucose uptake inhibitors and promoters A tissue construct not containing vascular endothelial cells was prepared in the same manner as in Test Example 10, and evaluation was performed using the tissue construct 7 days after its formation. The sCMF used was prepared using a mixture of fragments of pig skin-derived collagen type I sponge and fragments of pig skin-derived collagen type III sponge.

[0166] In Test Example 10 (evaluation of glucose uptake), the DMEM medium containing NBD-modified glucose was supplemented with 125 μM of apigenin, a glucose uptake inhibitor, or 10 μg / mL of insulin, a glucose uptake promoter. The tissue was incubated and fluorescence measurements were performed in the same manner as in Test Example 10 at 20, 45, 70, 90, and 135 minutes.

[0167] The results are shown in Figure 16. The number of samples for each treatment was 5 (n = 5), and the glucose uptake in the control medium, the same as in Test Example 10, which did not contain either apigenin or insulin, was set at 100%. It was found that the addition of apigenin inhibited the amount of glucose uptake, while the addition of insulin increased the amount of glucose uptake. Furthermore, when both apigenin and insulin were added, the amount of glucose uptake increased, but the increase was smaller than when insulin was added alone, suggesting that the increase in glucose uptake was controlled by the concentration of each drug.

[0168] Test Example 12: Evaluation of the effects of glucose and fatty acid uptake promoters A tissue construct not containing vascular endothelial cells was prepared in the same manner as in Test Example 10, and evaluation was performed using the tissue construct 7 days after its formation. The sCMF used was prepared using a mixture of fragments of pig skin-derived collagen type I sponge and fragments of pig skin-derived collagen type III sponge.

[0169] Fluorescence measurements were performed 5, 30, and 60 minutes after incubation of the tissues in the same manner as in Test Example 10, except that 4 μM BODIPY-modified fatty acid (Invitrogen® BODIPY® 500 / 510 C1, C12 (4,4-Difluoro-5-Methyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid), Thermo Fisher Scientific) was added to the DMEM medium instead of the NBD-modified glucose in Test Example 10. Furthermore, 10 μg / mL of insulin was added to the DMEM medium containing the above-mentioned 4 μM BODIPY-modified fatty acid, and fluorescence measurements were performed in the same manner.

[0170] The results are shown in Figure 17. Graph (b) in Figure 17 shows the change in fatty acid uptake in Test Example 12. The number of samples for each treatment was 5 (n = 5), and the fatty acid uptake in the control medium, which was the same as that in Test Example 10 but did not contain insulin, was set at 100%. These results demonstrate that insulin promotes not only glucose uptake but also fatty acid uptake.

[0171] Graph (a) in Figure 17 shows the change in glucose uptake in Test Example 11. Fluorescence measurements were performed 20, 45, 70, and 90 minutes after incubation of the tissues. There were five samples (n = 5) for each treatment, and the glucose uptake in the control medium, the same as in Test Example 10, which did not contain insulin, was set at 100%. Comparing graphs (a) and (b) suggests that fatty acids may saturate their uptake more quickly than glucose.

[0172] <Test Example 13: Evaluation of the effect of fatty acid uptake inhibitors> Cell structures containing vascular endothelial cells and tissue structures not containing vascular endothelial cells were prepared in the same manner as in Test Example 10, and evaluation was carried out using the cell structures and tissue structures on the 14th day after their formation. sCMF prepared using fragments of type I collagen sponge derived from pig skin was used.

[0173] Instead of the NBD-modified glucose used in Test Example 10 (evaluation of glucose uptake), DMEM medium containing 4 μM BODIPY-modified fatty acid was supplemented with 100 ng / mL of TNFα, a fatty acid uptake inhibitor. Cell structures containing vascular endothelial cells and tissue structures not containing vascular endothelial cells were incubated, and fluorescence measurements were performed at 0, 5, 30, and 60 minutes in the same manner as in Test Example 10. The number of samples for each treatment was 5 (n=5), and the fatty acid uptake in the same control medium as in Test Example 10 but without TNFα was set to 100%.

[0174] The results are shown in Figure 18. Graph (b) in Figure 18 shows the results for a cell structure containing vascular endothelial cells, while graph (a) shows the results for a tissue structure not containing vascular endothelial cells. It was found that the addition of TNFα suppressed the uptake of fatty acids.

[0175] Test Example 14: Evaluation of glucose and fatty acid release and evaluation of the effect of fatty acid release promoters A tissue was prepared in the same manner as the cell construct of Test Example 10, except that vascular endothelial cells were not included, and evaluation was carried out using the tissue 14 days after its formation.

[0176] To measure the amount of glucose released after uptake, the cells were incubated for 60 minutes in DMEM medium supplemented with 0.1 mg / mL NBD-modified glucose, as in Test Example 10, and then the medium was replaced with 300 μL of DMEM medium supplemented with NBD-modified glucose or 300 μL of DMEM medium supplemented with 2 mM isoproterenol and NBD-modified glucose. The cells were then incubated for 30 minutes to release glucose from the tissue, and fluorescence measurements were performed.

[0177] Similarly, to measure the amount of fatty acid released after uptake, the cells were incubated for 60 minutes in DMEM medium supplemented with 4 μM BODIPY-modified fatty acid, as in Test Example 12, and then replaced with 300 μL of DMEM medium supplemented with BODIPY-modified fatty acid or 300 μL of DMEM medium supplemented with 2 mM isoproterenol and BODIPY-modified fatty acid. The cells were then incubated for 30 minutes to release the fatty acid from the tissue, and fluorescence measurements were performed.

[0178] The results are shown in Figure 19. Graph (a) shows a comparison of the amount of glucose released, and graph (b) shows a comparison of the amount of fatty acid released. There were five samples (n = 3) for each treatment. The fluorescence intensity of the tissues in isoproterenol-free medium was set at 100%, and the fluorescence intensity of the tissues in isoproterenol-containing medium was compared to determine the percentage of the uptake that was released. Note that in the case of isoproterenol-free medium, it is thought that the uptake would not change significantly after 30 minutes of incubation.

[0179] When the medium was replaced with one containing isoproterenol, the amount of glucose or fatty acids taken up after 30 minutes of incubation was found to be reduced, indicating that the addition of isoproterenol promoted the release of both glucose and fatty acids.

Claims

1. A method for evaluating the effect of a drug that inhibits or promotes adipose tissue metabolism using a cell structure, comprising: the cell structure comprises fragmented collagen and cells; A cell structure having a vascular network between cells, the cells including at least adipocytes and vascular endothelial cells, The method, wherein the fragmented collagen comprises a defibrated collagen component having an average length of 5 μm or more and 30 μm or less.

2. 2. The method of claim 1, wherein the average length of the defibrated collagen component is between 6.6 μm and 23 μm.

3. an administration step of administering a drug that inhibits or promotes adipose tissue metabolism to the cell structure; an evaluation step of evaluating the effect of the drug based on metabolic changes in the cell structure to which the drug is administered; 3. The method of claim 1 or 2, comprising:

4. The evaluation step Altered glucose and / or fatty acid uptake, and / or Changes in the release of incorporated glucose and / or fatty acids The method of claim 3, comprising evaluating the following as an index:

5. A method for screening for a drug that inhibits or promotes adipose tissue metabolism using a cell structure, comprising: the cell structure comprises fragmented collagen and cells; A cell structure having a vascular network between cells, the cells including at least adipocytes and vascular endothelial cells, The fragmented collagen contains a defibrated collagen component having an average length of 5 μm or more and 30 μm or less. method.

6. The method of claim 5, wherein the average length of the defibrated collagen component is between 6.6 μm and 23 μm.

7. Measuring metabolism in the cell structure to which the drug has been administered; A step of comparing the metabolism in the cell structure to which the drug has been administered with the metabolism in the cell structure to which the drug has not been administered, and selecting the drug as a candidate drug for inhibiting adipose tissue metabolism if the metabolism in the cell structure to which the drug has been administered is lower, or Measuring metabolism in the cell structure to which the drug has been administered; The method of claim 5 or 6, further comprising the steps of: comparing the metabolism in a cell structure to which a drug has been administered with the metabolism in a cell structure to which the drug has not been administered; and, if the metabolism in the cell structure to which the drug has been administered is higher, selecting the drug as a candidate drug for promoting the metabolism of adipose tissue.

8. The method according to claim 7, wherein the comparison of metabolism in the cell structure is carried out using the uptake of glucose and / or fatty acids and / or the release of the taken-up glucose and / or fatty acids as indicators.

9. The method according to any one of claims 1 to 7, wherein the cell structure is a cell structure that has aggregated into a mass without being attached to a support.

10. The method of claim 9, wherein the particles are substantially spherical.

11. The method according to any one of claims 1 to 10, wherein the vascular network is formed between the adipocytes in the cell structure.

12. The method according to any one of claims 1 to 11, wherein in the cell structure, the adipocytes comprise mature adipocytes.

13. The method according to any one of claims 1 to 12, 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.

14. The method according to any one of claims 1 to 13, wherein the cell structure further comprises fibrin.

15. The method according to any one of claims 1 to 14, wherein the cell structure is for transplantation.

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