Three-dimensional mammary gland model and manufacturing method thereof

Abstract

A three-dimensional mammary gland model comprising a three-dimensional tissue comprising cells and fragmented extracellular matrix components, the cells comprising mature adipocytes and mammary gland epithelial cells.

Description

Technical Field

[0001] The invention relates to a three-dimensional breast model and a manufacturing method thereof. Background Art

[0002] Various studies have been conducted on the preparation of mammary models (non-patent documents 1 to 2 and patent document 1). Non-patent document 1 discloses a mammary model in which fibroblasts and mammary epithelial cells are embedded in matrix gel. Non-patent document 2 discloses a mammary model using primary mouse mammary organoids cultured in matrix gel. Patent document 1 discloses a cell culture method in which mammary epithelial cells are cultured in a multilayered state in a divided microscopic space to obtain a tissue structure with biological similar functions.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: International Publication No. 2009 / 099153

[0006] Non-patent literature

[0007] Non-Patent Literature 1: Zuzana Koledova ed., “3D Cell Culture: Methods and Protocols”, Methods in Molecular Biology, vol. 1612.

[0008] Non-Patent Literature 2: Sumbal J, et al., “Primary Mammary Organoid Model of Lactation and Involution.”, Front Cell Dev Biol. 2020 Mar 19; 8: 68. Summary of the Invention

[0009] Problems to be solved by the invention

[0010] However, milk cannot be obtained from the mammary tissue model described in Non-Patent Document 1 and the tissue structure obtained using mammary epithelial cells described in Patent Document 1. In addition, the method for producing the tissue structure described in Patent Document 1 requires the use of a special cell culture vessel, which is not simple. Casein, one of the milk proteins, was detected in the mammary model described in Non-Patent Document 2, but this could not be reproduced using cells other than primary mouse mammary organoids. As such, it is difficult to create a mammary model that differentiates mammary epithelial cells to the point where milk can be obtained in vitro.

[0011] The present invention aims to provide a three-dimensional mammary gland model capable of obtaining milk. In addition, the present invention aims to provide a method for easily and conveniently manufacturing the three-dimensional mammary gland model.

[0012] Means for solving problems

[0013] That is, the present invention includes, for example, the following inventions. [1]

[0015] A three-dimensional mammary gland model is composed of a three-dimensional tissue body comprising cells and fragmented extracellular matrix components, wherein the cells include mature adipocytes and mammary epithelial cells. [2]

[0017] The three-dimensional mammary gland model according to [1] has a space surrounded by two layers of the above-mentioned mammary epithelial cell layers. [3]

[0019] The three-dimensional mammary gland model according to [1] or [2], wherein, in the two mammary epithelial cell layers, the mammary epithelial cells on the lumen side are CK8 / 18-positive cells, and the mammary epithelial cells on the basement membrane side are CK14-positive cells. [4]

[0021] The three-dimensional mammary gland model according to any one of [1] to [3], wherein the fragmented extracellular matrix component contains fragmented collagen. [5]

[0023] The three-dimensional breast model according to any one of [1] to [4], wherein the three-dimensional tissue further comprises a hydrogel. [6]

[0025] The three-dimensional breast model according to [5], wherein the hydrogel is a fibrin gel. [7]

[0027] A method for producing a three-dimensional mammary gland model comprises: contacting cells with a fragmented extracellular matrix component in an aqueous medium; and culturing the cells after contact with the fragmented extracellular matrix component; wherein the cells include mature adipocytes, adipose-derived stem cells, and mammary epithelial cells. [8]

[0029] The production method according to [7], wherein the fragmented extracellular matrix component contains fragmented collagen. [9]

[0031] The production method according to [7] or [8], comprising the step of further contacting a hydrogel precursor in the contacting step, and gelling the hydrogel precursor after the contacting step and before the culturing step.

[10]

[0033] The production method according to [9], wherein the hydrogel is a fibrin gel.

[0034] Effects of the Invention

[0035] According to the present invention, a three-dimensional mammary gland model capable of obtaining milk can be provided. In addition, according to the present invention, a method for easily producing the three-dimensional mammary gland model can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic diagram showing a space surrounded by two layers of mammary epithelial cells.

[0037] Figure 2 The image (A) shows the results of CK8 / 18 immunostaining of a three-dimensional tissue section in Experimental Example 1. Figure 2 A partially enlarged image of (A) (B), an image showing the results of CK14 immunostaining (C), and Figure 2 (D) A magnified image of a portion of (C).

[0038] Figure 3 These are images showing the results of Nile red staining and CK8 / 18 immunostaining of the three-dimensional tissue in Experimental Example 1.

[0039] Figure 4 The images (A) and (B) show the results of Nile red staining of the three-dimensional tissue of Example 2 in Test Example 2 and the results of Nile red staining of the three-dimensional tissue of Comparative Example 1, respectively.

[0040] Figure 5 This is a graph comparing the fluorescence intensity of Nile red staining of three-dimensional tissues in Experimental Example 2.

[0041] Figure 6 The images (A) and (B) show the results of Western blotting analysis of casein in three-dimensional tissues in Experimental Example 3 and the results of Western blotting analysis of casein in two-dimensionally cultured mammary epithelial cells, respectively.

[0042] Figure 7 This is a graph showing the results of quantifying the band brightness of casein in three-dimensional tissues and two-dimensionally cultured mammary epithelial cells measured by Western blotting in Experimental Example 3.

[0043] Figure 8 This is a graph showing the results of analysis of CLDN3 expression in three-dimensional tissues in Experimental Example 4.

[0044] Figure 9 This is a diagram schematically showing an example of the mechanism leading to milk secretion in the mammary gland. DETAILED DESCRIPTION

[0045] Hereinafter, the embodiment for implementing the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0046] 〔3D Breast Model〕

[0047] The three-dimensional mammary gland model of this embodiment is composed of a three-dimensional tissue comprising cells and fragmented extracellular matrix components. The cells include mature adipocytes and mammary epithelial cells. In the three-dimensional mammary gland model of this embodiment, the differentiation of mammary epithelial cells is promoted, and milk can be obtained.

[0048] The three-dimensional breast model of this embodiment is a breast model that can be artificially created in vitro and does not include breast tissue obtained solely by isolation from a living organism. The structure of breast tissue in a living organism is more complex than that of a three-dimensional tissue. Therefore, the three-dimensional breast model of this embodiment can easily distinguish between a three-dimensional tissue and the breast tissue of a living organism.

[0049] In this specification sheets, " three-dimensional tissue body " refers to the aggregate (massive cell group) of the cell that cell is three-dimensionally configured via extracellular matrix component and / or fragmentation extracellular matrix component, is the aggregate made by cell culture.The shape of three-dimensional tissue body is not particularly limited, for example, sheet, spheroid, roughly spheroid, ellipsoid, roughly ellipsoid, hemispherical, roughly hemispherical, semicircular, roughly semicircular, cuboid, roughly cuboid etc. can be enumerated.From the viewpoint of the mammary gland closer to organism, the shape of three-dimensional tissue body is preferably spheroid or roughly spheroid.

[0050] The total number of cells comprising the three-dimensional tissue of this embodiment is not particularly limited and can be appropriately determined taking into account the thickness and shape of the constructed three-dimensional mammary gland model, the size of the cell culture vessel used for construction, and the like. Furthermore, the total number of cells comprising the three-dimensional tissue of this embodiment has the same meaning as the total number of cells comprising the three-dimensional mammary gland model of this embodiment.

[0051] In this specification, 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 cell's origin is also not particularly limited and may be a somatic cell derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, or other sources, 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.

[0052] In this specification, "stem cell" refers to a cell with self-replication ability and multi-differentiation ability. Stem cells include pluripotent stem cells with the ability to differentiate into any cell type and tissue stem cells (also referred to as somatic stem cells) with the ability to differentiate into a specific cell type. As pluripotent stem cells, for example, embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells) and artificial pluripotent stem cells (iPS cells) can be mentioned. As tissue stem cells, for example, mesenchymal stem cells (such as adipose-derived stem cells, bone marrow-derived stem cells), hematopoietic stem cells and neural stem cells can be mentioned. As adipose-derived stem cells (ADSC), for example, human adipose-derived stem cells and bovine adipose-derived stem cells can be mentioned.

[0053] The three-dimensional tissue of this embodiment contains at least mature adipocytes and mammary epithelial cells as cells.

[0054] Mature adipocytes are cells included within the concept of adipocytes, and can be identified by, for example, the size of their fat droplets. Fat droplets are intracellular organelles that store lipids such as triglycerides (neutral fat) and cholesterol. These lipids are covered by a phospholipid monolayer membrane, resulting in a droplet-like shape. Furthermore, proteins unique to adipose tissue (such as perilipin) can be observed on the surface of these phospholipids. While the size of mature adipocyte droplets varies, for example, a mature adipocyte can be identified if the average droplet size is 20 μm or larger.

[0055] Mature adipocytes may be, for example, cells collected from subcutaneous adipose tissue, epicardial adipose tissue, or the like, cells obtained by inducing differentiation from collected cells, or cells artificially differentiated from stem cells. While mature adipocytes are not particularly limited, in order to use the three-dimensional organoid of this embodiment as a mammary gland model, mature adipocytes derived from mammary gland adipose tissue are preferably used.

[0056] The content of mature adipocytes relative to the total number of cells in the three-dimensional tissue can be, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more, and can be 95% or less, 90% or less, 80% or less, or 75% or less. Furthermore, to achieve greater suitability as a mammary gland model, the content of mature adipocytes relative to the total number of cells in the three-dimensional tissue in this embodiment can be 30% or more and 80% or less, 35% or more and 75% or less, or 40% or more and 65% or less.

[0057] The cells in the three-dimensional tissue of this embodiment may include undifferentiated adipocytes such as adipose-derived stem cells. The content of adipose-derived stem cells relative to the total number of cells in the three-dimensional tissue may be, for example, 1% or more, 3% or more, 5% or more, 10% or more, or 15% or more, and may be 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less.

[0058] Mammary epithelial cells are cells present in the mammary gland, and are, for example, cells that have functions such as milk synthesis and secretion. Furthermore, mammary epithelial cells accumulate fat droplets as they differentiate. Mammary epithelial cells that accumulate fat droplets have differentiated to the point where they can secrete milk. Therefore, the mammary epithelial cells in the three-dimensional tissue of this embodiment can be mammary epithelial cells that accumulate fat droplets.

[0059] Mammary epithelial cells may be, for example, primary mammary epithelial cells collected from the mammary gland of an animal, cells obtained by culturing primary mammary epithelial cells, cultured cell lines obtained by isolating primary mammary epithelial cells, or mammary epithelial cells obtained by artificially differentiating from stem cells.

[0060] The mammary epithelial cell content relative to the total number of cells in the three-dimensional tissue can be, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more, and can be 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less. Furthermore, from the perspective of being more suitable as a mammary gland model and from the perspective of facilitating the formation of a space surrounded by two mammary epithelial cell layers described later, the mammary epithelial cell content relative to the total number of cells in the three-dimensional tissue in this embodiment can be 15% or more and 70% or less, 15% or more and 35% or less, or 20% or more and 30% or less.

[0061] In the three-dimensional tissue of the present embodiment, the ratio (number of cells) of mature adipocytes to mammary epithelial cells is not particularly limited, and may be, for example, 0.25:1 to 2.5:1, or 1:1 to 2.4:1.

[0062] The cells may further include cells other than mature adipocytes and mammary epithelial cells. Cells other than mature adipocytes and mammary epithelial cells include fibroblasts (e.g., human mammary fibroblasts (HMF), human skin-derived fibroblasts (NHDF), human cardiac fibroblasts (NHCF), human gingival fibroblasts (HGF), etc.), adipocytes other than mature adipocytes and adipose-derived stem cells, vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVEC), etc.), and cancer cells (e.g., human breast cancer cells (MCF7, MDA-MB-453), etc.).

[0063] "Fragmented extracellular matrix component" can be obtained by fragmenting the extracellular matrix component. The extracellular matrix component is an aggregate of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. Extracellular matrix molecules can be substances present in the extracellular space in multicellular organisms. As extracellular matrix molecules, any substance can be used as long as the growth of cells and the formation of cell aggregates are not adversely affected. As extracellular matrix molecules, collagen, laminin, fibronectin, pornectin, elastin, cell adhesion protein, nidogen, fibrillin and proteoglycans etc. can be enumerated, but are not limited to these. As extracellular matrix components, these extracellular matrix molecules can be used alone or in combination.

[0064] Extracellular matrix molecules can be modified or mutant forms of the above-mentioned extracellular matrix molecules, or they can be polypeptides such as chemically synthesized peptides. Extracellular matrix molecules can have repetitions of the sequence represented by Gly-XY, a characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent an arbitrary amino acid residue. Multiple Gly-XY residues can be the same or different. The repetition of the sequence represented by Gly-XY reduces constraints on the configuration of the molecular chain. In extracellular matrix molecules having repetitions of the sequence represented by Gly-XY, the proportion of the sequence represented by Gly-XY in the total amino acid sequence can be 80% or more, preferably 95% or more. Alternatively, the extracellular matrix molecule can be a polypeptide having an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). Examples of the extracellular matrix molecules containing a sequence represented by Gly-XY and an RGD sequence include collagen, fibronectin, bonectin, laminin, and cadherin.

[0065] As collagen, for example, fibrous collagen and non-fibrous collagen can be mentioned. Fibrous collagen refers to collagen that becomes the main component of collagen fibers, specifically, type I collagen, type II collagen, type III collagen, etc. can be mentioned. As non-fibrous collagen, for example, type IV collagen can be mentioned. Collagen is preferably fibrous collagen.

[0066] Examples of proteoglycans include chondroitin sulfate proteoglycans, heparin sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans, but are not limited thereto.

[0067] The extracellular matrix component can include at least one selected from collagen, laminin and fibronectin, and from the viewpoint of excellent cell adhesion, preferably includes collagen. Collagen is preferably fibrous collagen, more preferably type I collagen. Fibrous collagen can use commercially available collagen, and as its specific example, can enumerate the pigskin-derived type I collagen of Japan Ham Co., Ltd. system.

[0068] The extracellular matrix component can be an extracellular matrix component of animal origin. As the animal species that becomes the source of the extracellular matrix component, for example, humans, pigs, cattle, etc. can be enumerated, but are not limited to these. The extracellular matrix component can use a component of one animal origin, or can use the component of multiple animal origins in combination.

[0069] In this manual, " fragmentation " refers to making the aggregate of extracellular matrix molecules into smaller size. Fragmentation can be carried out under the condition of cutting off the bond in extracellular matrix molecules, or it can be carried out under the condition of not cutting off the bond in extracellular matrix molecules. The extracellular matrix component through fragmentation can include the composition obtained by applying physical force that above-mentioned extracellular matrix component is defibrinated, i.e. the extracellular matrix component (defibrinated extracellular matrix component) obtained by defibrination. Defibrination is a mode of fragmentation, for example, is carried out under the condition of not cutting off the bond in extracellular matrix molecules.

[0070] As the method by extracellular matrix component fragmentation, there is no particular restriction.As the method extracellular matrix component is carried out to fibrillation, for example, can apply physical power to extracellular matrix component by ultrasonic homogenizer, stirring homogenizer, and high pressure type homogenizer etc. to fibrillate.When using stirring homogenizer, extracellular matrix component can be directly homogenized, also can be homogenized in aqueous media such as physiological saline.In addition, by adjusting the time, number of times etc. that homogenize, can obtain the fibrillation extracellular matrix component of millimeter size, nanometer size.Fibrillation extracellular matrix component also can be carried out fibrillation and obtain by repeatedly carrying out freeze thawing.

[0071] The fragmented extracellular matrix component may contain a fibrillating extracellular matrix component in at least a portion. The fragmented extracellular matrix component may be composed solely of a fibrillating extracellular matrix component. That is, the fragmented extracellular matrix component may be a fibrillating extracellular matrix component. The fibrillating extracellular matrix component preferably contains a fibrillating collagen component. The fibrillating collagen component preferably maintains the triple helical structure of the collagen source. The fibrillating collagen component may be a component that completely or partially maintains the triple helical structure of the collagen source.

[0072] As the shape of the fragmented extracellular matrix component, for example, fibrous can be enumerated. Fibrous refers to the shape consisting of filamentous extracellular matrix components, or the shape consisting of filamentous extracellular matrix components in intermolecular crosslinking. At least a portion of the fragmented extracellular matrix component can be fibrous. The fibrous extracellular matrix component comprises a plurality of filamentous extracellular matrix molecules assembled and formed filaments (thin fibers), thin fibers further assembled and formed filaments, and the composition obtained by defibrating these filaments. In the fibrous extracellular matrix component, the RGD sequence is not destroyed and is preserved.

[0073] The average length of the fragmented extracellular matrix component can be more than 100 nm and less than 400 μm, or more than 100 nm and less than 200 μm. In one embodiment, the average length of the fragmented extracellular matrix component can be more than 5 μm and less than 400 μm, can be more than 10 μm and less than 400 μm, can be more than 22 μm and less than 400 μm, or can be more than 100 μm and less than 400 μm. In another embodiment, the average length of the fragmented extracellular matrix component can be less than 100 μm, can be less than 50 μm, can be less than 30 μm, can be less than 15 μm, can be less than 10 μm, can be less than 1 μm, and can be more than 100 nm. In the entire fragmented extracellular matrix component, the average length of most of the fragmented extracellular matrix components can be within the above numerical range. Specifically, the average length of 95% of the fragmented extracellular matrix components in the entire fragmented extracellular matrix component can be within the above numerical range. The fragmented extracellular matrix component may be a fragmented collagen component having an average length within the above range, or may be a defibrillated collagen component having an average length within the above range.

[0074] The average diameter of the fragmented extracellular matrix component can be 10 nm or more and 30 μm or less, 30 nm or more and 30 μm or less, 50 nm or more and 30 μm or less, 100 nm or more and 30 μm or less, 1 μm or more and 30 μm or less, 2 μm or more and 30 μm or less, 3 μm or more and 30 μm or less, 4 μm or more and 30 μm or less, or 5 μm or more and 30 μm or less. The fragmented extracellular matrix component is preferably a fragmented collagen component having an average diameter within the above range, and more preferably a defibrillated collagen component having an average diameter within the above range.

[0075] The average length and average diameter of the fragmented extracellular matrix components can be determined by measuring each fragmented extracellular matrix component using an optical microscope and performing image analysis. In this specification, "average length" refers to the average value of the length of the sample in the longitudinal direction, and "average diameter" refers to the average value of the length of the sample in a direction perpendicular to the longitudinal direction.

[0076] The fragmented extracellular matrix component may, for example, include a fragmented collagen component or may be composed of a fragmented collagen component. A "fragmented collagen component" refers to a component obtained by fragmenting a collagen component such as a fibrous collagen component, which maintains a triple helical structure. The average length of the fragmented collagen component is preferably 100 nm to 200 μm, more preferably 22 μm to 200 μm, and further 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 further preferably 20 μm to 30 μm.

[0077] At least a portion of the fragmented extracellular matrix component may be cross-linked between molecules or within molecules. The extracellular matrix component may be cross-linked within or between molecules of the extracellular matrix molecules constituting the extracellular matrix component.

[0078] As the method for cross-linking, for example, physical cross-linking by applying heat, ultraviolet rays, radiation, etc., chemical cross-linking by cross-linking agents, enzyme reactions, etc. can be enumerated, and the method is not particularly limited. From the viewpoint of not hindering cell growth, physical cross-linking is preferred. Cross-linking (physical cross-linking and chemical cross-linking) can be cross-linking via covalent bonds.

[0079] In the case where the extracellular matrix component comprises collagen composition, crosslinking can be formed between collagen molecules (triple helical structure), can also be formed between the collagen fibrils formed by collagen molecules.Crosslinking can be the crosslinking (thermal crosslinking) performed by heat.Thermal crosslinking can be implemented by using a vacuum pump to carry out heat treatment under reduced pressure.In the case of carrying out the thermal crosslinking of collagen composition, the extracellular matrix component can be crosslinked by the amino group of collagen molecules and the carboxyl group of same or other collagen molecules to form peptide bond (NH-CO).

[0080] The extracellular matrix component can also be cross-linked by using a cross-linking agent. Cross-linking agent can be, for example, a cross-linking agent that can cross-link a carboxyl group and an amino group or a cross-linking agent that can cross-link an amino group to each other. As a cross-linking agent, from the viewpoint of economy, safety and operability, for example, preferably aldehydes, carbodiimides, epoxides and imidazoles cross-linking agents, specifically, water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide sulfonate can be enumerated.

[0081] The quantitative degree of cross-linking can be appropriately selected based on the type of extracellular matrix components, the means of cross-linking, etc. The degree of cross-linking can be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, and can be 30% or less, 20% or less, or 15% or less. By keeping the degree of cross-linking within the above range, the extracellular matrix molecules can be appropriately dispersed, and the redispersibility after drying and storage is good.

[0082] In the case where the amino group in the extracellular matrix component is used for cross-linking, the degree of cross-linking can be quantitatively determined based on the TNBS (trinitrobenzene sulfonic acid) method. The degree of cross-linking obtained by the TNBS method can be within the above range. The degree of cross-linking obtained by the TNBS method is the ratio of the amino group in the amino group possessed by the extracellular matrix that is used for cross-linking. In the case where the extracellular matrix component comprises a collagen component, the degree of cross-linking preferably measured by the TNBS method is within the above range.

[0083] The degree of cross-linking can be calculated by quantifying the carboxyl groups. For example, in the case of water-insoluble extracellular matrix components, quantification can be performed by the TBO (Toluidine Blue O) method. The degree of cross-linking obtained by the TBO method can also be within the above range.

[0084] The content of the extracellular matrix component in the three-dimensional tissue body can be 0.01 to 90 mass%, preferably 10 to 90 mass%, preferably 10 to 80 mass%, preferably 10 to 70 mass%, preferably 10 to 60 mass%, preferably 1 to 50 mass%, preferably 10 to 50 mass%, more preferably 10 to 30 mass%, and more preferably 20 to 30 mass%, based on the above-mentioned three-dimensional tissue body (dry weight).

[0085] Here, the "extracellular matrix components in a three-dimensional tissue" refers to the extracellular matrix components constituting the three-dimensional tissue, and may be derived from endogenous extracellular matrix components or exogenous extracellular matrix components.

[0086] "Endogenous extracellular matrix components" refer to extracellular matrix components produced by extracellular matrix-producing cells. Examples of extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and adipocytes. Endogenous extracellular matrix components may be fibrous or non-fibrous.

[0087] "Exogenous extracellular matrix components" refer to extracellular matrix components supplied from the outside. The three-dimensional tissue of this embodiment contains fragmented extracellular matrix components as exogenous extracellular matrix components. The exogenous extracellular matrix components may be the same as or different from the endogenous extracellular matrix components of the animal species from which they are derived. Examples of the animal species from which they are derived include humans, pigs, and cattle. In addition, the exogenous extracellular matrix components may also be artificial extracellular matrix components.

[0088] That is, when the three-dimensional tissue body contains endogenous extracellular matrix components and fragmented extracellular matrix components, the extracellular matrix component content of the three-dimensional tissue body refers to the total amount of the endogenous extracellular matrix components and the fragmented extracellular matrix components. The extracellular matrix content can be calculated based on the volume of the obtained three-dimensional tissue body and the mass of the decellularized three-dimensional tissue body.

[0089] 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" representing the collagen component supplied from the outside is an aggregate of collagen molecules formed by multiple collagen molecules, specifically, fibrous collagen, non-fibrous collagen, etc. Exogenous collagen components are preferably fibrous collagen. The above-mentioned fibrous collagen refers to the collagen component that becomes the main component of collagen fibers, for example, type I collagen, type II collagen, and type III collagen can be mentioned. The above-mentioned fibrous collagen can use commercially available collagen, and as a specific example thereof, type I collagen derived from pigskin made by Ham Co., Ltd. of Japan can be mentioned. As an exogenous non-fibrous collagen, for example, type IV collagen can be mentioned.

[0090] In the exogenous extracellular matrix component, the animal species derived from can be different from the cell. In addition, when the cell comprises an extracellular matrix-producing cell, with regard to the exogenous extracellular matrix component, the animal species derived from can be different from the extracellular matrix-producing cell. That is, the exogenous extracellular matrix component can be a heterologous extracellular matrix component.

[0091] For example, when the extracellular matrix component contained in the three-dimensional tissue body is a collagen component, as a method for quantifying the amount of collagen component in the three-dimensional tissue body, for example, the following method for quantifying hydroxyproline can be cited. Hydrochloric acid (HCl) is mixed with a lysate in which the three-dimensional tissue body is dissolved, and after incubation at high temperature for a predetermined time, the solution is returned to room temperature, and the supernatant after centrifugation is diluted to a predetermined concentration to prepare a sample. After the hydroxyproline standard solution is treated in the same manner as the sample, it is diluted in stages to prepare a standard solution. The sample and the standard solution are respectively treated with a hydroxyproline analysis 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 that of the standard solution. It should be noted that the three-dimensional tissue body can also be directly suspended in a high concentration of hydrochloric acid, and the dissolved solution can be centrifuged to recover the supernatant for quantitative determination of the collagen component. In addition, the three-dimensional tissue body to be dissolved can be in a state of being directly recovered from the culture medium, or it can be dried after recovery and dissolved in a state where the liquid component is removed. However, when dissolving a three-dimensional tissue body recovered directly from the culture medium to quantify the collagen component, it is expected that the measured value of the three-dimensional tissue weight will deviate due to the influence of the culture medium components absorbed by the three-dimensional tissue body and the culture medium residue caused by problems with the experimental technique. Therefore, from the perspective of stably measuring the weight of the structure and the amount of collagen component per unit weight, it is preferred to use the weight after drying as the basis.

[0092] More specifically, for example, the following method can be used as a method for quantifying the amount of collagen components.

[0093] (Sample Preparation)

[0094] The total volume of freeze-dried three-dimensional tissues was mixed with 6 mol / L HCl and incubated at 95°C for at least 20 hours in a heating block, then returned to room temperature. After centrifugation at 13,000 g for 10 minutes, the supernatant of the sample solution was recovered. For the assay described below, samples were prepared by diluting 200 μL of the sample solution with 100 μL of ultrapure water after appropriate dilution with 6 mol / L HCl to ensure that the results fell within the range of the standard curve. A 35 μL sample was used.

[0095] (Preparation of Standards)

[0096] To a screw-cap tube, add 125 μL of a standard solution (1200 μg / mL, acetic acid solution) and 125 μL of 12 mol / L HCl, mix, incubate at 95°C for 20 hours in a heating block, and then return to room temperature. After centrifugation at 13,000 g for 10 minutes, dilute the supernatant with ultrapure water to prepare 300 μg / mL S1. S1 is then diluted in stages to prepare S2 (200 μg / mL), S3 (100 μg / mL), S4 (50 μg / mL), S5 (25 μg / mL), S6 (12.5 μg / mL), and S7 (6.25 μg / mL). Also prepare S8 (0 μg / mL) with only 90 μL of 4 mol / L HCl.

[0097] (analyze)

[0098] Add 35 μL of standard and sample to a plate (attached to the QuickZyme Total Collagen Assay kit, QuickZyme Biosciences). Add 75 μL of analysis buffer (attached to the above kit) to each well. Seal the plate with a seal, shake for 20 minutes, and incubate at room temperature. Peel off the seal and add 75 μL of detection reagent (reagent A: B = 30 μL: 45 μL, attached to the above kit) to each well. Seal the plate with a seal, mix the solution by shaking, and incubate at 60°C for 60 minutes. Cool thoroughly on ice, peel off the seal, and measure the absorbance at 570 nm. Calculate the amount of collagen component by comparing the absorbance of the sample with that of the standard.

[0099] The collagen component shared in the three-dimensional tissue body can also be specified by its area ratio or volume ratio. "Specify by area ratio or volume ratio" means, for example, that the collagen component in the three-dimensional tissue body is distinguishable from other tissue components by known staining methods (e.g., immunostaining using anti-collagen antibodies, or Masson's trichrome staining), etc., and then, using naked eye observation, various microscopes and image analysis software, etc., the ratio of the area of ​​the collagen component shared in the three-dimensional tissue body as a whole is calculated. When specified by area ratio, it is not limited to what kind of cross section or surface in the three-dimensional tissue body is used to specify the area ratio. For example, when the three-dimensional tissue body is a spheroid, etc., it can be specified by a cross-sectional view passing through its approximate center.

[0100] For example, when the collagen component in a three-dimensional tissue is specified by an area ratio, the ratio of its area is 0.01 to 99%, preferably 1 to 99%, preferably 5 to 90%, preferably 7 to 90%, preferably 20 to 90%, and more preferably 50 to 90%, based on the area of ​​the entire three-dimensional tissue. As described above, the "collagen component in a three-dimensional tissue" is the same as above. The ratio of the area of ​​the collagen component constituting the three-dimensional tissue refers to the ratio of the area of ​​the endogenous collagen component and the exogenous collagen component combined. The ratio of the area of ​​the collagen component can be calculated, for example, by staining the obtained three-dimensional tissue with Masson's trichrome and calculating the ratio of the area of ​​the collagen component stained blue to the total area of ​​a cross section passing through the approximate center of the three-dimensional tissue.

[0101] The three-dimensional tissue of this embodiment has a space surrounded by two layers of the mammary epithelial cells. Figure 1 This figure shows a space surrounded by two layers of mammary epithelial cells in one embodiment. As the mammary epithelial cells in the three-dimensional tissue of one embodiment differentiate, they form a circular layer consisting of mammary epithelial cells 1 on the lumen side (inner side) and a circular layer consisting of mammary epithelial cells 2 on the basement membrane side (outer side). This forms a space 3 surrounded by two layers of mammary epithelial cells.

[0102] Regarding the mammary epithelial cells 1 and 2 in the gap 3 surrounded by the two layers of mammary epithelial cell layers, for example, the mammary epithelial cells 1 on the lumen side are preferably CK8 / 18 positive cells, and the mammary epithelial cells 2 on the basement membrane side are CK14 positive cells. In this specification, "positive cells" refer to cells that express specific marker proteins on the cell surface or in the cell. For example, "CK8 / 18 positive cells" refer to cells that express CK8 protein or CK18 protein. CK8 / 18 and CK14 are differentiation markers that can be confirmed in the mammary tissue of an organism. Such a two-layer structure is also observed in the mammary gland of an organism. Therefore, compared with this situation, it is more suitable as a mammary gland model.

[0103] The three-dimensional tissue structure of this embodiment may further include a hydrogel. In this specification, "hydrogel" refers to a substance composed of polymers cross-linked through hydrogen bonds, ionic bonds, coordination bonds, covalent bonds, and other methods to form a three-dimensional network structure, with a liquid such as water contained within the three-dimensional network structure. When the three-dimensional tissue structure of this embodiment includes a hydrogel, its contraction is promoted, resulting in a spherical shape similar to that of breast tissue in a living organism.

[0104] As hydrogel, for example, fibrin gel, collagen gel, gelatin gel, hyaluronic acid gel, alginate gel, pectin gel etc. can be mentioned, but are not limited to these. The hydrogel is preferably a fibrin gel. Fibrin is a component generated by thrombin acting on fibrinogen and releasing A chain and B chain from the N-terminus of Aα chain and Bβ chain. Fibrin is formed by contacting fibrinogen with thrombin. The hydrogel can be composed of one of the above, or it can be combined to include two or more.

[0105] When the three-dimensional tissue of this embodiment includes a hydrogel, the three-dimensional tissue may be unembedded or embedded in the hydrogel, but is preferably embedded from the perspective of further promoting contraction. Embedding the three-dimensional tissue with a hydrogel means that the hydrogel is present in at least a portion or all of the intercellular spaces outside the three-dimensional tissue or between the outside and inside.

[0106] The three-dimensional tissue body of this embodiment is obtained by co-culturing mature adipocytes and mammary epithelial cells. As the mammary epithelial cells differentiate, they are capable of accumulating a large number of fat droplets within the three-dimensional tissue body. As used herein, "capable of accumulating a large number of fat droplets" means that, in an image obtained by microscopic observation of fat droplets in a three-dimensional tissue body stained with a fluorescent substance capable of detecting fat droplets, such as Nile red, the area of ​​the region containing fat droplets can be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more, and can be 95% or less, 90% or less, 85% or less, 80% or less, or 75% or less. The area ratio of the region containing fat droplets is not limited to a specific cross-section or surface within the three-dimensional tissue body; for example, in the case of a spherical tissue body, the area ratio can be defined using a cross-section through its approximate center.

[0107] In the three-dimensional organoid of this embodiment, due to the progress of mammary epithelial cell differentiation, differentiation markers such as CK8 / 18 and CK14, which are identified in mammary tissue in living organisms, are expressed in the three-dimensional organoid. In the three-dimensional organoid of this embodiment, when immunostained for CK8 / 18 or CK14 and observed under a microscope, the area of ​​the region where CK8 / 18 or CK14 is identified can be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more relative to the area of ​​the three-dimensional organoid, and can be 95% or less, 90% or less, 85% or less, 80% or less, or 75% or less.

[0108] As described above, the three-dimensional breast model of this embodiment is capable of producing milk. "Producing milk" means detecting milk proteins contained in milk in the three-dimensional tissue that constitutes the three-dimensional breast model. "Detecting milk proteins" may mean, for example, an amount of milk protein that is 1.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times greater than the amount of milk protein in two-dimensionally cultured mammary epithelial cells.

[0109] The amount of milk protein in the three-dimensional tissue is not particularly limited as long as it can be measured. For example, it can be measured by the method described in the Examples below, more specifically, by Western blotting or the like.

[0110] Examples of milk proteins include casein, lactoferrin, lactalbumin, and lactoglobulin. The milk proteins detected in the three-dimensional tissue of the present embodiment may be one or more of the above.

[0111] The three-dimensional tissue of this embodiment may also be responsive to a substance that stimulates milk secretion. "Responsiveness to a substance that stimulates milk secretion" may mean an increase in the amount of milk protein compared to a state without milk secretion stimulation, and may also mean an increase in the amount of milk protein of, for example, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, or 1.5-fold or more.

[0112] Examples of substances that stimulate milk secretion include prolactin and oxytocin.

[0113] For example, after the three-dimensional tissue of this embodiment is cultured in a culture medium containing 1 μg / mL of prolactin for 7 days, when casein in the three-dimensional tissue is detected by protein blotting, the brightness of the casein band relative to α-tubulin can be detected to be greater than 1%, greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%, and can be detected to be less than 300%, less than 200%, or less than 100%.

[0114] For example, when the three-dimensional organoids of this embodiment are cultured for 7 days in a medium containing 1 μg / mL of prolactin and the expression level of Claudin 3 (CLDN3) in the three-dimensional organoids is analyzed by quantitative PCR, the expression level of CLDN3 in the three-dimensional organoids can be increased by 1.05-fold or more, 1.1-fold or more, 1.15-fold or more, 1.2-fold or more, 1.25-fold or more, or 1.3-fold or more, compared to the expression level of CLDN3 in the three-dimensional organoids cultured for 7 days in a medium not containing prolactin. CLDN3 is one of the four transmembrane proteins, claudin, that constitute tight junctions, a type of intercellular binding.

[0115] The thickness of the three-dimensional tissue body can be 10 μm or more, 30 μm or more, 50 μm or more, 100 μm or more, 300 μm or more, or 1000 μm or more. Such a three-dimensional tissue body has a structure that is closer to that of biological tissue and is suitable as a substitute for experimental animals and a transplant material. The upper limit of the thickness of the three-dimensional tissue body is not particularly limited, and for example, it can be 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, 1 mm or less, 300 μm or less, 200 μm or less, 150 μm or less, or 100 μm or less.

[0116] Here, the "thickness of a three-dimensional tissue body" refers to the distance between the two ends in a direction perpendicular to the main surface when the three-dimensional tissue body is a rectangular parallelepiped. When the main surface has irregularities, the thickness refers to the distance at the thinnest part of the main surface.

[0117] When the three-dimensional tissue body is spherical or substantially spherical, the thickness of the three-dimensional tissue body refers to the diameter of the three-dimensional tissue body. When the three-dimensional tissue body is ellipsoidal or substantially ellipsoidal, the thickness of the three-dimensional tissue body refers to the minor diameter of the three-dimensional tissue body. When the three-dimensional tissue body is substantially spherical or substantially ellipsoidal and has uneven surfaces, the thickness refers to the shortest distance between two points where a straight line passing through the center of gravity of the three-dimensional tissue body intersects the surface.

[0118] The three-dimensional tissue of the present embodiment is constructed in a cell culture container. As the cell culture container, there is no particular limitation as long as it is a container that can construct a three-dimensional tissue and can culture the constructed three-dimensional tissue. As the cell culture container, specifically, culture dishes, cell culture plug-ins (such as Transwell (registered trademark) plug-in, Netwell (registered trademark) plug-in, Falcon (registered trademark) cell culture plug-in, Millicell (registered trademark) cell culture plug-in, etc.), test tubes, flasks, bottles, flat plates, etc. can be mentioned. In the construction of the three-dimensional tissue, from the viewpoint of being able to more appropriately perform the evaluation using the three-dimensional tissue, culture dishes or various cell culture plug-ins are preferred.

[0119] [Method for producing a three-dimensional breast model]

[0120] The method for producing a three-dimensional mammary gland model of this embodiment includes: a step of contacting cells with fragmented extracellular matrix components in an aqueous medium (contact step); and a step of culturing the cells after contact with the fragmented extracellular matrix components (culturing step). The cells include mature adipocytes, adipose-derived stem cells, and mammary epithelial cells, as described above.

[0121] The method for producing a three-dimensional mammary gland model of this embodiment has the above-described configuration, and therefore can co-culture mammary gland epithelial cells and mature adipocytes, thereby promoting the differentiation of mammary gland epithelial cells and easily producing a mammary gland tissue model capable of producing milk.

[0122] In the contacting step, mature adipocytes, adipose-derived stem cells, mammary epithelial cells, and fragmented extracellular matrix components are brought into contact in an aqueous medium.

[0123] The ratio (cell number) of mature adipocytes to mammary epithelial cells in the contact step is as described above. Furthermore, the ratio (cell number) of mature adipocytes to adipose-derived stem cells in the contact step can be 1:1 to 2.5:1, or 1.2:1 to 2:1. Furthermore, the ratio (cell number) of adipose-derived stem cells to mammary epithelial cells in the contact step can be 1:0.5 to 1:4, 1:0.5 to 1:3, 1:0.5 to 1:2, 1:0.5 to 1:1, or 1:1 to 1:2.

[0124] "Aqueous medium" refers to a liquid having water as an essential component. As an aqueous medium, there is no particular limitation as long as it is a medium in which fragmented extracellular matrix components can stably exist. As a specific example of an aqueous medium, for example, as an aqueous medium, liquid culture media such as physiological saline such as phosphate buffered saline (PBS), Dulbecco's Modified Eagle medium (DMEM), and mammary epithelial cell culture medium (MEGM) can be cited, but are not limited thereto. The liquid culture medium can be a mixed culture medium having two or more culture media.

[0125] Examples of methods for contacting cells with fragmented extracellular matrix components include a method of mixing an aqueous medium containing fragmented extracellular matrix components with a culture medium containing cells, a method of adding an aqueous medium containing fragmented extracellular matrix components to a culture medium containing cells, a method of adding cells to an aqueous medium containing fragmented extracellular matrix components, and a method of separately adding fragmented extracellular matrix components and cells to a pre-prepared aqueous medium.

[0126] The order in which cells are contacted with the fragmented extracellular matrix components is not particularly limited. For example, a portion of the cells may be contacted with the fragmented extracellular matrix components before the remaining cells are contacted with the fragmented extracellular matrix components. Alternatively, all cells may be contacted with the fragmented extracellular matrix components simultaneously or substantially simultaneously. From the perspective of facilitating the production of three-dimensional tissues, it is preferred that mature adipocytes be contacted with the fragmented extracellular matrix components after adipose-derived stem cells and mammary epithelial cells are contacted with the fragmented extracellular matrix components.

[0127] The aqueous medium containing cells and fragmented extracellular matrix components may or may not be mixed by stirring after the addition of each substance. The contacting step may include a step of contacting the cells and fragmented extracellular matrix components and then incubating for a predetermined time.

[0128] The concentration of the fragmented extracellular matrix components in the contact step can be appropriately determined based on the shape and thickness of the target three-dimensional tissue, the size of the culture vessel, etc. For example, the concentration of the fragmented extracellular matrix components in the aqueous medium in the contact step can be 0.1 to 90% by mass, or 1 to 30% by mass.

[0129] The amount of the fragmented extracellular matrix component in the contact step is, for example, relative to 1.0×10 6 The amount of cells of cells can be 0.1-100 mg, 0.5-50 mg, 0.8-25 mg, 1.0-10 mg, 1.0-5.0 mg, 1.0-2.0 mg, or 1.0-1.8 mg, can be 0.7 mg or more, 1.1 mg or more, 1.2 mg or more, 1.3 mg or more, or 1.4 mg or more, and can 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.

[0130] In the contact step, the mass ratio of the fragmented extracellular matrix component to the cells (fragmented extracellular matrix component / cell) is preferably 1 / 1 to 1000 / 1, more preferably 9 / 1 to 900 / 1, and even more preferably 10 / 1 to 500 / 1.

[0131] In the culturing step, the cells contacted with the fragmented extracellular matrix components are cultured to form a three-dimensional tissue.

[0132] Examples of the culture medium used in the culture step include mammary epithelial cell culture medium (e.g., MEGM medium (manufactured by Lonza)) and KBM medium. The culture medium may be serum-supplemented or serum-free. The culture medium may be supplemented with growth factors, hormones such as insulin, and the like. The culture medium may be a mixed medium containing two culture media.

[0133] The culture temperature in the culture step can be, for example, 20° C. to 40° C., or 30° C. to 37° C. The pH of the culture medium can be 6 to 8, or 7.2 to 7.4. The culture time can be 1 day to 2 weeks, or 1 to 2 weeks.

[0134] The culture vessel (support) is not particularly limited, and for example can be a culture dish, a well plug-in, a low-adhesion flat plate, or a flat plate with a bottom surface shape such as a U-character or a V-character. The above-mentioned cells can be cultured in a state where they adhere to the support, or they can be cultured without adhering to the support, or they can be cultured separately from the support midway in cultivation. In the case where the above-mentioned cells are cultured without adhering to the support or in the case where they are cultured separately from the support midway in cultivation, it is preferred to use a flat plate or a low-adsorption flat plate with a bottom surface shape such as a U-character or a V-character that inhibits the adhesion of cells to the support.

[0135] The cell density in the culture medium during the culture step can be appropriately determined according to the shape and thickness of the target three-dimensional tissue, the size of the culture vessel, etc. For example, the cell density in the culture medium during the culture step can be 1 to 10 8 cells / mL, or 10 3 ~10 7 In addition, the cell density in the culture medium in the culturing step may be the same as the cell density in the aqueous medium in the contacting step.

[0136] The production method of this embodiment may include a step (gelation step) of further contacting a hydrogel precursor in the contact step, and gelling the hydrogel precursor after the contact step and before the culturing step.

[0137] Hydrogel precursor refers to a substance that forms a hydrogel under certain external stimuli such as chemical stimulation or physical stimulation, and is a substance in a state of not forming a hydrogel. As a hydrogel, the above-mentioned hydrogel can be enumerated. As a hydrogel precursor, for example, fibrinogen, collagen, gelatin, hyaluronic acid, alginic acid and pectin can be enumerated. As a hydrogel precursor, it is preferred to contain fibrinogen. Fibrinogen forms a fibrin gel by reacting with thrombin. Therefore, as long as fibrinogen and thrombin contact in the aqueous medium in the contact process or gelation process, for example, when the above-mentioned aqueous medium contains serum, owing to containing thrombin in the serum, even if thrombin is not added specifically, a fibrin gel can be formed.

[0138] The concentration of the hydrogel precursor in the aqueous medium during the contact step may be 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, 0.5 mg / mL or more, 0.6 mg / mL or more, 0.7 mg / mL or more, 0.8 mg / mL or more, 0.9 mg / mL or more, 1.0 mg / mL or more, or 2.0 mg / mL or more, based on the total amount of the aqueous medium. The concentration of the hydrogel precursor in the aqueous medium may be 10.0 mg / mL or less, 8.0 mg / mL or less, 7.0 mg / mL or less, 6.0 mg / mL or less, 5.0 mg / mL or less, 3.0 mg / mL or less, 1.0 mg / mL or less, 0.8 mg / mL or less, or 0.6 mg / mL or less, based on the total amount of the aqueous medium.

[0139] For example, when the hydrogel precursor contains two or more substances, such as a combination of fibrinogen and thrombin, the contacting step can be performed by mixing a first solution containing cells, a fragmented extracellular matrix component, an aqueous medium, and a first hydrogel precursor (e.g., fibrinogen) with a second solution containing an aqueous medium and a second hydrogel precursor (e.g., thrombin). It should be noted that the second solution may contain cells and / or fragmented extracellular matrix components, and both the first solution and the second solution may contain cells and / or fragmented extracellular matrix components.

[0140] In the case where the hydrogel precursor is further contacted in the contacting step, the order in which the cells, fragmented extracellular matrix components, and hydrogel precursors are contacted is not particularly limited. From the viewpoint of easily producing a three-dimensional tissue body, the contacting step is preferably performed after adipose-derived stem cells and mammary epithelial cells are contacted with fragmented extracellular matrix components, and then mature adipocytes and hydrogel precursors are contacted with fragmented extracellular matrix components. In the case where the hydrogel precursor comprises two or more substances, it is preferred that adipose-derived stem cells, mammary epithelial cells, fragmented extracellular matrix components, and mature adipocytes are contacted with the first hydrogel precursor in the same order, and then the first hydrogel precursor is contacted with the second hydrogel precursor.

[0141] The gelation step is a step of forming a hydrogel containing cells and fragmented extracellular matrix components. The gelation step may include a step of incubating for a predetermined time. The gelation step may be a step of embedding cells and fragmented extracellular matrix components in the hydrogel.

[0142] Example

[0143] Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.

[0144] Cells and reagents used in the production of the three-dimensional breast model are shown in Tables 1-2 below. In addition, the production method of CMF and the collection methods of adipose-derived human stem cells and mature adipocytes are described below.

[0145] Table 1 Cells

[0146]

[0147] Table 2 Culture media, reagents, and various experimental equipment

[0148]

[0149]

[0150] <Preparation of CMF>

[0151] Add a neutralization buffer mixed with 2.125 mL of 0.05 N NaOH and 2.125 mL of 10×PBS to 17 mL of a 3 mg / mL collagen solution, and heat it at 37 °C for 30 minutes to gelify it. Freeze-dry the obtained gel for 72 hours to obtain freeze-dried collagen after gelification. Add 15 mL of 85 v / v% ethanol to the freeze-dried collagen after gelification, and defibrillate it for 6 minutes using a homogenizer (Probe: S10N-10G-ST shaft generator). Centrifuge the sample obtained after defibrillation at 10,000 rpm, discard the supernatant, add 15 mL of 70 v / v% ethanol respectively, and perform suction and blowing using a homogenizer (S10N-8G shaft generator). Centrifuge the sample obtained after suction and blowing at10,000 rpm, discard the supernatant, add 15 mL of ultrapure water, and perform suction and blowing. Then, perform ultrasonic treatment 10 times for 20 seconds using an ultrasonic crusher. Freeze-dry the sample containing defibrillated collagen (CMF) obtained through the above operations in a state where CMF is dispersed in water.

[0152] <Collection of mature adipocytes and ADSCs>

[0153] Wash human adipose tissue fragments with PBS containing 5% antibiotics. Divide 4-6g of tissue into 6 wells of a 6-well plate. Use scissors and tweezers to finely cut into a size of approximately 1-3mm in 2mL of 2mg / mL collagenase solution. After incubation at 37°C and 230rpm for 30 minutes, blow and aspirate with a 10mL pipette. After a further incubation of 30 minutes, add 2ml of DMEM to each well to stop the collagenase reaction. Filter the lysate with a mesh filter with a pore size of 500μm and centrifuge at 80g for 3 minutes at room temperature (15-25°C). Mature adipocytes are contained in the upper yellow oily layer, and adipose-derived stem cells and blood cells are contained in the pellet. Use a 10mL pipette to aspirate the medium between the upper and lower layers and discard it. Wash the mature adipocytes contained in the upper layer and the adipose-derived stem cells and blood cells contained in the lower layer twice with 25mL of PBS (5% BSA, 1% antibiotics). During washing, centrifugation was performed in the same manner as above to separate the cells into three layers: an upper layer, a lower layer, and a medium between the upper and lower layers. The medium between the upper and lower layers was aspirated and discarded. After washing twice, the cells were washed with 25 mL of DMEM.

[0154] Only the upper layer containing mature adipocytes was collected and dispensed into Eppendorf tubes. Nuclei were stained with Hoechst (1000-fold diluted Hoechst, 15 minutes staining), and the number of cells was counted using a Turk Burk hemocytometer under a fluorescence microscope.

[0155] The ADSC pellet was suspended in 10 mL of DMEM and seeded into a 10 cm culture dish for subculture. The ADSCs were detached from the culture dish using trypsin / EDTA, suspended in 1 mL of DMEM, and the cell number was counted.

[0156] [Test Example 1: Morphological Confirmation by Immunostaining in a Three-Dimensional Mammary Gland Model]

[0157] <Fabrication of 3D Breast Model>

[0158] Recover the previously cultured hADSCs and HMECs, calculate the required number of cells after counting the number of cells. Measure the required amount of CMF into a microtube, add 1 ml of Milli-Q, pipette to disperse the CMF, and centrifuge to remove the supernatant. Add the cell suspension of hADSCs and HMECs with the calculated cell number to a microtube containing CMF, centrifuge at 3500 rpm for 1 minute at room temperature without pipetting, and remove the supernatant. Next, add the required amount of fibrinogen to the microtube and mix without bubbling. Then, take the required amount of mature adipocytes and add them to the microtube, gently mix, and prepare a cell suspension of each cell, CMF, and fibrinogen at the concentration shown in Table 3. 5 μL of the prepared cell suspension (the amount of 1 tissue portion shown in Table 3) is dispensed into a 96-well round-bottom plate. Then, incubate at 37°C and 5% CO2 for 15 minutes to form a fibrin gel. 100 μL of DMEM was added to each well to allow the gel containing various cells and CMF to float. The floated gel was scooped out with a spatula and transferred to a 24-well plate containing 500 μL of MEGM medium. The medium was replaced every 2-3 days for 7 days to produce a 3D mammary gland model composed of the 3D organoid described in Example 1.

[0159] Table 3

[0160] / 1ml (cell suspension) 5μL (1 tissue portion) mature adipocytes (mAD) <![CDATA[3.5×10 6 cells]]> <![CDATA[1.75×10 4 cells]]> Adipose-derived stem cells (hADSCs) <![CDATA[3.0×10 6 cells]]> <![CDATA[1.5×10 4 cells]]> Mammary epithelial cells (HMECs) <![CDATA[3.0×10 6 cells]]> <![CDATA[1.5×10 4 cells]]> CMF 12 mg (1.2%) 0.06 mg (1.2%) Fibrinogen 6.6mg 0.33mg

[0161] <Fixation and Immunostaining of 3D Organoids>

[0162] The three-dimensional organoids of Example 1 were fixed and immunostained in the following order. The three-dimensional organoids of Example 1 after 7 days of culture were scooped out with a medicine spoon and placed in a microtube containing 500 μL of PBS. The PBS was removed from the microtube and PBS was added again. This was repeated three times to wash the three-dimensional organoids. After washing with PBS, 500 μL of 4% paraformaldehyde was added and the mixture was allowed to stand overnight. Then, the paraformaldehyde was removed and the three-dimensional organoids were washed three times with PBS. 100 μL of each of 0.05% Triton X-100 and PBS was added to the microtube and allowed to stand at room temperature for 15 minutes. The supernatant was removed, and the three-dimensional organoids were washed three times with PBS, followed by addition of 1% BSA-PBS and allowed to stand at room temperature for 60 minutes. The anti-CK8 / 18 antibody or anti-CK14 antibody as the primary antibody was diluted 100-fold with 1% BSA-PBS, and 100 μL of each was added to the microtube and allowed to stand at 4°C overnight. After washing the three-dimensional organoids three times with PBS, secondary antibodies (anti-Alexa Fluor (trademark) 546-conjugated goat anti-rabbit IgG secondary antibody or anti-Alexa Fluor (trademark) 647-conjugated goat anti-mouse IgG secondary antibody, 1 / 200 dilution) diluted in 1% BSA-PBS, Hoechst (1 / 1000 dilution), and Nile Red (1 / 1000 dilution) were added and allowed to stand at room temperature for 2 hours. After washing the three-dimensional organoids three times with PBS, the tissues were observed using a confocal laser scanning microscope FV3000.

[0163] Figure 2 (A) shows the observation results of the cross section of the three-dimensional tissue of Example 1 by CK8 / 18 staining. Figure 2 (C) shows the observation results of the cross section of the three-dimensional tissue of Example 1 by CK14 staining. Figure 2 (B) or Figure 2 (D) are Figure 2 (A) or Figure 2 Figure (C) is a part of the amplification. In the three-dimensional tissue of embodiment 1, it was observed that the differentiation markers expressed in the mammary gland of the organism, i.e., CK8 / 18 and CK14, were expressed. In addition, the three-dimensional tissue of embodiment 1 has a space surrounded by two layers of mammary epithelial cell layers, which are formed by the mammary epithelial cells on the lumen side as CK8 / 18 positive cells and the mammary epithelial cells on the basement membrane side as CK14 positive cells. Therefore, the three-dimensional tissue of embodiment 1 includes the mammary epithelial cells after differentiation, and shows that it is an organoid closer to the mammary gland of the organism.

[0164] It should be noted that similar results were obtained even when a mixed medium containing 10 μM insulin and suitable for MEGM and KBM medium for vascular endothelial cell culture was used as the culture medium.

[0165] Figure 3 The results of observation of the three-dimensional organoid of Example 1 based on Nile Red staining (left) and CK8 / 18 staining (right) are shown. Fat droplets are found within the three-dimensional organoid of Example 1 and also within the mammary epithelial cells. This indicates that the mammary epithelial cells in the three-dimensional organoid of Example 1 are differentiated.

[0166] It should be noted that the same results were obtained when a three-dimensional tissue was produced by the same method as the three-dimensional tissue in Example 1, except that mature adipocytes, adipose-derived stem cells, and mammary epithelial cells were used at a ratio of 1.2:1:0.5 (cell number).

[0167] [Test Example 2: Comparison of Fat Droplets in a Three-Dimensional Breast Model Based on Nile Red Staining]

[0168] <Fabrication of 3D Breast Model>

[0169] A three-dimensional mammary gland model composed of the three-dimensional tissue body of Example 2 was obtained by the same method as the three-dimensional tissue body of Example 1. A three-dimensional mammary gland model composed of the three-dimensional tissue body of Comparative Example 1 was obtained by the same method as the three-dimensional tissue body of Example 1, except that no CMF was used.

[0170] <Fixation and Immunostaining of 3D Organoids>

[0171] In the same manner as in Test Example 1, the three-dimensional tissues of Example 2 and Comparative Example 1 were stained with Nile Red.

[0172] Figure 4 The results of observation of the three-dimensional tissues of Example 2 and Comparative Example 1 using Nile Red are shown. Figure 5 This is a graph showing the fluorescence intensity of Nile red in the three-dimensional tissue bodies of Example 2 and Comparative Example 1. The three-dimensional tissue body of Example 2 has a higher fluorescence intensity of Nile red than the three-dimensional tissue body of Comparative Example 1. This indicates that the three-dimensional tissue body of Example 2 is more capable of accumulating fat droplets than the three-dimensional tissue body of Comparative Example 1.

[0173] [Test Example 3: Evaluation of the Expression Amount of Milk Protein Secreted from a Three-Dimensional Mammary Gland Model]

[0174] <Fabrication of 3D Breast Model>

[0175] By the same method as that for the three-dimensional tissue body of Example 1, a three-dimensional mammary gland model composed of the three-dimensional tissue body of Example 3 was obtained.

[0176] <Quantification of Milk Protein>

[0177] The three-dimensional organoids of Example 3 were cultured in MEGM medium containing 1 μg / mL prolactin for 7 days to stimulate milk secretion. Then, casein, a milk protein, was detected in the three-dimensional organoids of Example 3. Furthermore, as a control, casein was also detected in the three-dimensional organoids of Example 3 cultured in MEGM medium without prolactin for 7 days. Furthermore, 5×10 4 The same experiment was performed on mammary epithelial cells cultured in two dimensions for 7 days.

[0178] In the three-dimensional tissue body of Example 3 or the mammary epithelial cells cultured in two dimensions, casein was detected by protein blotting in the following order. After culturing in MEGM medium containing prolactin for 7 days, NucleoSpin (registered trademark) RNA / Protein was used to extract protein from the three-dimensional tissue body of Example 3 or the mammary epithelial cells cultured in two dimensions according to the experimental protocol of the product. Prepare an electrophoresis tank and gel, install the gel in the electrophoresis tank, fill the electrophoresis tank with 1×SDS buffer, and clean the gel. Apply each sample to the gel in such a way that the total protein amount contained in each sample reaches about 10μg, and perform electrophoresis at 150-200V for about 40 minutes. After the electrophoresis is completed, wash the gel with milli-Q and immerse it in 1× transfer buffer for 15 minutes. Immerse the membrane in 100% ethanol for about 10 minutes, and place the membrane hydrophilized with ethanol in transfer buffer and let it stand for more than 5 minutes. Set it in the transfer box in the order of transfer stack, membrane, gel, and transfer stack. Tilt the transfer box, remove excess buffer, and then transfer. After transfer, wash the membrane with 1×TBS-T and block with blocking buffer for 1 hour. After blocking, wash the membrane with 1×TBS-T, immerse the membrane in primary antibody (anti-casein antibody or anti-α-tubulin antibody) diluted with 1×TBS-T, and incubate at 4°C overnight. Then, wash the membrane with 1×TBS-T, immerse the membrane in secondary antibody (goat anti-rabbit StarBright Blue 700 antibody or Alexa Fluor (trademark) 488-conjugated goat anti-rat IgG secondary antibody) diluted with 1×TBST for 1 hour. Wash the membrane with 1×TBST, detect the bands with an imager, and digitize the brightness of the bands using ImageJ.

[0179] Figure 6 These images show the results of Western blotting detection of casein in the three-dimensional organoids or two-dimensionally cultured mammary epithelial cells of Example 3. Figure 7This is a graph showing the numerical band brightness of casein by Western blotting in the three-dimensional organoids of Example 3 or mammary epithelial cells cultured in two dimensions. Even when prolactin was used to stimulate milk secretion, casein could not be detected in the mammary epithelial cells cultured in two dimensions. In contrast, casein was detected in the three-dimensional organoids of Example 3 even without prolactin-based stimulation of milk secretion. Furthermore, it can be seen that when prolactin-based stimulation of milk secretion was performed, the amount of casein increased compared to when no stimulation of milk secretion was performed, indicating responsiveness to substances that stimulate milk secretion.

[0180] The above results show that the three-dimensional mammary gland model of the present invention can produce milk.

[0181] [Test Example 4: Gene Analysis in a Three-Dimensional Mammary Gland Model]

[0182] <Fabrication of 3D Breast Model>

[0183] By the same method as that for the three-dimensional tissue body of Example 1, a three-dimensional mammary gland model composed of the three-dimensional tissue body of Example 4 was obtained.

[0184] <Analysis of Expression Levels of Tight Junction Proteins>

[0185] The three-dimensional organoids of Example 4 were cultured for 7 days in MEGM medium containing 1 μg / mL prolactin to stimulate milk secretion. As a control, the three-dimensional organoids of Example 4 were cultured for 7 days in MEGM medium (without prolactin). The expression level of CLDN3, a protein that constitutes tight junctions, was then analyzed in the three-dimensional organoids of Example 4 according to the following procedure. CLDN3 expression was also analyzed in the control three-dimensional organoids.

[0186] RNA was extracted from a three-dimensional mammary gland model on day 7 of culture. RNA was extracted using the NucleoSpin RNA / Protein Kit (Model 740933, manufactured by Machery-Nagel). The extracted RNA was reverse transcribed to synthesize complementary DNA (cDNA). cDNA was synthesized using the QuantiTec (registered trademark) Reverse Transcription Kit (Model 20513, manufactured by QIAGEN). The synthesized cDNA was subjected to quantitative PCR analysis to analyze CLDN3 expression. Quantitative PCR analysis was performed using the Taqman (registered trademark) Fast Advanced Master Mix (Model 4444556, manufactured by Thermo Fisher Scientific) and the Taqman (registered trademark) Gene Expression assay (Model 4331182, manufactured by Thermo Fisher Scientific). As primers for quantitative PCR, oligonucleotides with Assay ID: Hs00265816_S1 were used for CLDN3. As an analysis apparatus, the StepOnePlus Real-Time PCR System (manufactured by Thermo Fisher Scientific) was used, and analysis was performed according to the thermal profile recommended by the manufacturer.

[0187] Figure 8 Graph showing the results of analyzing the expression level of CLDN3 in the three-dimensional mammary gland model of Example 4. In the three-dimensional mammary gland model of Example 4, it was confirmed that the expression level of CLDN3 tends to increase in response to prolactin. Figure 9 As shown, it is known that in the normal milk secretion mechanism of the mammary gland, when the expression level of CLDN3 responds to the hormone stimulation brought by prolactin ( Figure 9 When the mammary epithelial cells in the glandular chamber rise, tight junctions are formed to prevent the transport of substances between cells ( Figure 9 (2)). When tight junctions are formed, casein, lactose, and milk fat droplets are formed and released into the lumen ( Figure 9 (3)). Therefore, it can be seen that in this embodiment, the fact that the expression level of CLDN3 increases in response to prolactin suggests the possibility that the three-dimensional mammary gland model can mimic the mechanism of milk secretion in vivo.

[0188] Description of Reference Numerals

[0189] 1 Mammary epithelial cells on the lumen side

[0190] 2 Mammary epithelial cells on the basement membrane side

[0191] 3 gaps

Claims

1. A three-dimensional mammary gland model, comprising a three-dimensional tissue body comprising cells and fragmented extracellular matrix components, The cells contained mature adipocytes and mammary epithelial cells. The three-dimensional mammary gland model according to claim 1 , comprising a space surrounded by two layers of mammary epithelial cells.

3. The three-dimensional breast model according to claim 1 or 2, wherein: In the two mammary epithelial cell layers, the mammary epithelial cells on the lumen side are CK8 / 18 positive cells, and the mammary epithelial cells on the basement membrane side are CK14 positive cells.

4. The three-dimensional breast model according to claim 1 or 2, wherein: The fragmented extracellular matrix component contains fragmented collagen.

5. The three-dimensional breast model according to claim 1 or 2, wherein: The three-dimensional tissue body further comprises a hydrogel.

6. The three-dimensional breast model according to claim 5, wherein: The hydrogel is a fibrin gel.

7. A method for manufacturing a three-dimensional breast model, comprising: a step of contacting cells with fragmented extracellular matrix components in an aqueous medium, and a step of culturing the cells after contacting with the fragmented extracellular matrix component; in, The cells contain mature adipocytes, adipose-derived stem cells and mammary epithelial cells.

8. The manufacturing method according to claim 7, wherein: The fragmented extracellular matrix component contains fragmented collagen.

9. The production method according to claim 7 or 8, comprising the following steps: In the contacting step, the hydrogel precursor is further contacted. The hydrogel precursor is gelled after the contacting step and before the culturing step.

10. The manufacturing method according to claim 9, wherein: The hydrogel is a fibrin gel.

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