WO2025211450A1 - Three-dimensional mammary gland model and method for producing same - Google Patents

Abstract

The present disclosure pertains to a method for producing a three-dimensional mammary gland model that comprises a three-dimensional tissue body including mature adipocytes and mammary epithelial cells, the method comprising: a step for gelling a mixture that contains a fragmented extracellular matrix component, an alginate gel precursor, and cells that include adipose-derived stem cells and mammary epithelial cells; and a step for subjecting the gelled mixture to differentiation induction culture to thereby differentiate at least a portion of the adipose-derived stem cells into the mature adipocytes to form the three-dimensional tissue body.

Description

Three-dimensional mammary gland model and its manufacturing method

The present invention relates to a three-dimensional mammary gland model and a method for manufacturing the same.

Various studies have been conducted on the creation of mammary gland models (Non-Patent Documents 1-2 and Patent Document 1). Non-Patent Document 1 discloses a mammary gland model in which fibroblasts and mammary epithelial cells are embedded in Matrigel. Non-Patent Document 2 discloses a mammary gland model using primary mouse mammary gland organoids cultured in Matrigel. Patent Document 1 discloses a cell culture method in which mammary epithelial cells are cultured in a stratified state in a compartmented microspace to obtain a tissue structure with biological functions.

International Publication No. 2009/099153

Zuzana Koledova ed., “3D CellCulture: Methods and Protocols”, Methods in Molecular Biology, vol. 1612, pp 107-124. Sumbal J, et al., “Primary Mammary Organoid Model of Lactation and Involution.”, Front Cell Dev Biol. 2020Mar 19;8:68.

Mature adipocytes extracted from living adipose tissue are used to create three-dimensional mammary gland models. However, because mature adipocytes are collected from surplus tissue during surgery, there was room for improvement in terms of the stability of the material supply.

The object of the present invention is to provide a method for producing a three-dimensional mammary gland model that closely resembles the structure of a living organism, without using mature adipocytes extracted from adipose tissue in the living organism, and a three-dimensional mammary gland model obtained by this method.

The present invention includes, for example, the following inventions.
[1]
A method for producing a three-dimensional mammary gland model consisting of a three-dimensional tissue structure containing mature adipocytes and mammary epithelial cells, the method comprising: a step of gelling a mixture containing cells including adipose-derived stem cells and mammary epithelial cells, fragmented extracellular matrix components, and an alginate gel precursor; and a step of inducing differentiation of the gelled mixture by culturing the mixture to differentiate at least a portion of the adipose-derived stem cells into the mature adipocytes, thereby forming the three-dimensional tissue structure.
[2]
The method according to [1], wherein the fragmented extracellular matrix components contain fragmented collagen components.
[3]
The method according to [2], wherein the average length of the fragmented collagen components is 100 nm to 200 μm.
[4]
The method according to [2] or [3], wherein the fragmented collagen component is homogenized in an aqueous medium.
[5]
The method according to any one of [1] to [4], wherein the differentiation-inducing culture is carried out by culturing the gelled mixture in a medium containing a fatty acid.
[6]
[5] The method according to [5], wherein the fatty acid is oleic acid.
[7]
The method according to any one of [1] to [6], further comprising, after the step of forming the three-dimensional tissue, a step of culturing the three-dimensional tissue in a medium containing a fatty acid.
[8]
The method according to any one of [1] to [7], wherein the mixture is gelled by contacting it with calcium ions.
[9]
The method according to any one of [1] to [8], wherein the content of the alginate gel precursor in the mixture is 0.20 w/v% or more and 1.0 w/v% or less, based on the total amount of the mixture.
[10]
The method according to any one of [1] to [9], wherein the content of the fragmented extracellular matrix components in the mixture is 0.1 w/v% or more and 0.8 w/v% or less, based on the total amount of the mixture.
[11]
A three-dimensional mammary gland model comprising a three-dimensional tissue structure containing cells including mature adipocytes and mammary epithelial cells, fragmented extracellular matrix components, and alginate gel.
[12]
The three-dimensional mammary gland model according to [11], wherein the three-dimensional mammary gland model has an acinar structure.
[13]
The three-dimensional mammary gland model according to [11] or [12], wherein the fragmented extracellular matrix components contain fragmented collagen components.
[14]
The three-dimensional mammary gland model according to any one of [11] to [13], wherein the three-dimensional tissue contains fatty acids.
[15]
The three-dimensional mammary gland model described in [14], wherein the fatty acid is oleic acid.
[16]
The three-dimensional mammary gland model according to any one of [11] to [15], constructed in a cell culture vessel.
[17]
A method for producing a three-dimensional mammary gland model consisting of a three-dimensional tissue containing mature adipocytes and mammary epithelial cells, the method comprising the steps of: gelling a mixture containing cells including adipose-derived stem cells and mammary epithelial cells, a fragmented collagen component, and an alginate gel precursor; and culturing the gelled mixture in a medium containing oleic acid to differentiate at least a portion of the adipose-derived stem cells into the mature adipocytes and form acinar structures in the three-dimensional tissue, wherein the content of the alginate gel precursor in the mixture is 0.20 w/v% or more and 0.30 w/v% or less, based on the total amount of the mixture, and the content of the fragmented collagen component is 0.1 w/v% or more and 0.8 w/v% or less, based on the total amount of the mixture.
[18]
A three-dimensional mammary gland model comprising a three-dimensional tissue body containing cells including mature adipocytes and mammary epithelial cells, fragmented extracellular matrix components, and alginate gel, and containing two or more aggregates of lipid droplets formed by aggregation of two or more of the mature adipocytes, each having a diameter of 20 μm or more.

The present invention provides a method for producing a three-dimensional mammary gland model that closely resembles the structure of a living organism, without using mature adipocytes extracted from the adipose tissue of the living organism, and a three-dimensional mammary gland model obtained by this method.

Figure 1 shows images of the observation results of a three-dimensional tissue constructed using a fragmented collagen component (CMF) and alginate gel, where (A) shows the results on day 7 of culture, and (B) shows the results on day 14 of culture. Figure 2 shows images of the three-dimensional tissue stained with Nile Red and DAPI (4',6-diamidino-2-phenylindole), where (A) shows the results without CMF and (B) shows the results with CMF. FIG. 3 shows images of the three-dimensional tissues stained with Nile red, DAPI, and CK8/18 immunostained. FIG. 4 shows images of the three-dimensional tissues stained with Nile Red and DAPI, where (A) shows the results when CMF was used, and (B) shows the results when non-fragmented collagen was used. Figure 5 shows images showing the observation results of a three-dimensional tissue in an example, where (A) shows the result when no CMF was included, (B) shows the result when the CMF content was 0.3%, and (C) shows the result when the CMF content was 0.6%. Figure 6 shows images showing the observation results of a three-dimensional tissue in an example, where (A) shows the result when no CMF was included, (B) shows the result when the CMF content was 0.9%, and (C) shows the result when the CMF content was 1.2%. FIG. 7 is an image showing the results of observation of a three-dimensional tissue in an example, showing the results of CK8 immunostaining and CK14 immunostaining of frozen sections of a three-dimensional tissue using ADSC or DFAT. FIG. 8 is an image showing the observation results of a three-dimensional tissue in an example, showing the results of immunostaining MFGE8 milk protein in frozen sections of a three-dimensional tissue using ADSC or DFAT. FIG. 9 is an image showing the observation results of a three-dimensional tissue in an example, showing the results of immunostaining of α-lactalbumin (LALBA) and butyrophilin (BTN1A1) in frozen sections of a three-dimensional tissue using ADSC or DFAT. FIG. 10 is a graph showing the results of quantifying the amount of α-lactalbumin in the three-dimensional tissue using an ELISA assay. Figure 11 shows images showing the results of Nile Red staining and DAPI staining of three-dimensional tissues in the examples, where (A) shows the results for a three-dimensional tissue prepared using CMF and alginate gel, (B) shows the results for a three-dimensional tissue prepared using alginate gel without using CMF and non-fragmented collagen, and (C) shows the results for a three-dimensional tissue prepared using non-fragmented collagen. Figure 12 shows images showing the results of bright-field observation of three-dimensional tissues in the examples, where (A) shows the results for a three-dimensional tissue prepared using CMF and alginate gel, and (B) shows the results for a three-dimensional tissue prepared using CMF and carrageenan. FIG. 13 is an image showing the fluorescence intensity ratio of Nile Red and DAPI in a three-dimensional tissue in an example. FIG. 14 shows images showing the results of Nile Red and DAPI immunostaining of a three-dimensional tissue structure in an example. FIG. 15 is a graph showing the count number of lipid droplets having an area value of 50 μm ² or more in a three-dimensional tissue in an example. FIG. 16 shows images showing the results of Nile Red staining and DAPI staining of three-dimensional tissues of examples in which the ratio of ADSC cell number to HMEC cell number (ADSC:HMEC) was changed. FIG. 17 is a schematic diagram illustrating a method for culturing the gelled mixture in the presence of fibroblasts. FIG. 18 shows the results of bright field observation of three-dimensional tissues in the examples after 0, 3, 5, 7, or 10 days of culture. FIG. 19 shows images of stained nuclei and F-actin in the three-dimensional tissue of the example, a merged image of these, and enlarged images of each image.

The following describes in detail the embodiments for implementing the present invention. However, the present invention is not limited to the following embodiments.

[Method for manufacturing a three-dimensional mammary gland model]
The method according to the present embodiment is a method for producing a three-dimensional mammary gland model consisting of a three-dimensional tissue structure containing mature adipocytes and mammary epithelial cells. The method includes the steps of: gelling a mixture containing cells including adipose-derived stem cells and mammary epithelial cells, fragmented extracellular matrix components, and an alginate gel precursor (gelling step); and culturing the gelled mixture to induce differentiation, thereby differentiating at least a portion of the adipose-derived stem cells into mature adipocytes, thereby forming a three-dimensional tissue structure (culturing step).

The method may further include a step (mixing step) of obtaining a mixture containing cells, fragmented extracellular matrix components, and an alginate gel precursor prior to the gelation step. Below, we will explain a method for producing a three-dimensional mammary gland model that includes the mixing step, gelation step, and culture step.

<Mixing process>
In the mixing step, cells including adipose-derived stem cells and mammary epithelial cells, fragmented extracellular matrix components, and an alginate gel precursor are mixed to obtain a mixture.

(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 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. The cell may be a stem cell, or a cultured cell such as a primary cultured cell, a subcultured cell, or a cell line cell.

As used herein, "stem cells" refers to cells 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 specific cell types. Examples of pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells). Examples of tissue stem cells include mesenchymal stem cells (e.g., adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells), hematopoietic stem cells, and neural stem cells.

The cells include at least adipose-derived stem cells and mammary epithelial cells. As used herein, "adipose-derived stem cells" encompasses adipose-derived mesenchymal stem cells (ADSCs) and dedifferentiated fat cells (DFAT).

Adipose-derived mesenchymal stem cells are mesenchymal stem cells collected from adipose tissue. Adipose-derived mesenchymal stem cells may be mesenchymal stem cells collected from, for example, subcutaneous adipose tissue or epicardial-derived adipose tissue. Adipose-derived mesenchymal stem cells are a group of cells obtained, for example, by adherent culturing a fraction (stromal fraction) of adipose tissue other than mature adipocytes.

Dedifferentiated adipocytes are stem cells that can be obtained by dedifferentiating mature adipocytes using ceiling culture. Mature adipocytes can be obtained by isolating them from adipose tissue. When the cells are adipose-derived stem cells that contain at least dedifferentiated adipocytes, milk proteins such as alpha-lactalbumin (LALBA), which plays an important role in lactose synthesis, which is essential for breast milk production, are produced more efficiently.

Adipose-derived stem cells may be derived from, for example, cattle, horses, mice, rats, or pigs. Examples of adipose-derived stem cells include human adipose-derived stem cells and bovine adipose-derived stem cells.

The ratio of the number of adipose-derived stem cells to the total number of cells may be 30% or more, 40% or more, 45% or more, or 50% or more, and may be 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less. The ratio of the number of adipose-derived stem cells to the total number of cells may be 30% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less, 40% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less, 45% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less, and 50% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less.

Mammary epithelial cells are cells present in the mammary gland and have functions such as the synthesis and secretion of milk. Mammary epithelial cells may be, for example, primary mammary epithelial cells collected from the mammary gland of an animal, cultured primary mammary epithelial cells, cultured cell lines established from primary mammary epithelial cells, or mammary epithelial cells artificially differentiated from stem cells.

The ratio of the number of mammary epithelial cells to the total number of cells may be 30% or more, 40% or more, 45% or more, or 50% or more, and may be 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less. The ratio of the number of mammary epithelial cells to the total number of cells may be 30% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less, 40% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less, 45% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less, and 50% or more and 90% or less, 80% or less, 70% or less, 60% or less, or 55% or less.

The ratio of the number of adipose-derived stem cells to the number of mammary epithelial cells (adipose-derived stem cells/mammary epithelial cells) may be 0.5 or greater, and from the viewpoint of making it easier to obtain a three-dimensional mammary gland model having an acinar structure, may be 0.6 or greater, 0.7 or greater, 0.8 or greater, or 0.9 or greater, and may be, for example, 5.0 or less, 4.5 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less. The ratio of the number of adipose-derived stem cells to the number of mammary epithelial cells may be 0.5 or greater and 5.0 or less, 4.5 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less. From the viewpoint of making it easier to obtain a three-dimensional mammary gland model having an acinar structure, the ratio of the number of adipose-derived stem cells to the number of mammary epithelial cells may be 0.6 or more and 5.0 or less, 4.5 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less, and may be 0.7 or more and 5.0 or less, 4.5 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less. It may be 0.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less; it may be 0.8 or more and 5.0 or less, 4.5 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less; it may be 0.9 or more and 5.0 or less, 4.5 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1.1 or less.

According to the method of this embodiment, a three-dimensional mammary gland model can be formed without using mature adipocytes in the mixing process, but mature adipocytes may be used if necessary.

Mature adipocytes are cells that fall under the concept of adipocytes, and can be identified, for example, using the size of the lipid droplets as an indicator. Lipid droplets are intracellular organelles that store lipids such as triglycerides (neutral fats) and cholesterol, and have a droplet-like shape because the lipids are covered by a single membrane of phospholipids. Proteins specific to adipose tissue (such as perilipin) are also expressed on the surface of the phospholipids. There is variation in the size of the lipid droplets in mature adipocytes, but for example, if the average lipid droplet size is 20 μm or larger, the cells can be considered mature adipocytes.

Mature adipocytes may be cells collected from, for example, subcutaneous adipose tissue, epicardial-derived adipose tissue, etc., or may be cells that have been induced to differentiate from collected cells, or may be cells artificially differentiated from stem cells.

The ratio of mature adipocytes to the total number of cells may be 10% or less, 5% or less, 3% or less, or 1% or less.

The cells may further include other cells in addition to adipose-derived stem cells, mammary epithelial cells, and mature adipocytes. Examples of other cells include fibroblasts (e.g., human mammary fibroblasts (HMF), human dermal fibroblasts (NHDF), human cardiac fibroblasts (NHCF), human gingival fibroblasts (HGF), etc.), mature adipocytes and adipocytes other than adipose-derived stem cells, vascular endothelial cells (e.g., human umbilical vein-derived endothelial cells (HUVEC)), and cancer cells (e.g., human breast cancer cells (MCF7, MDA-MB-453), etc.).

The ratio of the number of other cells to the total number of cells may be, for example, 10% or less, 5% or less, or 3% or less.

The cell concentration in the mixture may be 1 to 10 ⁸ cells/mL, and may be 10 ³ to 10 ⁷ cells/mL, based on the total volume of the mixture.

(Fragmented extracellular matrix components)
The mixture contains fragmented extracellular matrix components. By using the fragmented extracellular matrix components in a three-dimensional mammary gland model, a three-dimensional mammary gland model that more closely resembles the structure of a living body can be obtained. Specifically, by using the fragmented extracellular matrix components in a three-dimensional mammary gland model, a three-dimensional mammary gland model having an alveolar structure can be obtained.

"Fragmented extracellular matrix components" can be obtained by fragmenting extracellular matrix components. Extracellular matrix components are aggregates of extracellular matrix molecules formed by multiple extracellular matrix molecules. Extracellular matrix molecules may be substances that exist outside cells in multicellular organisms. Any substance can be used as an extracellular matrix molecule as long as it does not adversely affect cell growth or the formation of cell aggregates. Examples of extracellular matrix molecules include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycans. As an extracellular matrix component, one type of these extracellular matrix molecules may be used alone, or two or more types may be used in combination.

The extracellular matrix molecule may be a modified or variant of the above-mentioned extracellular matrix molecule, or may be a polypeptide such as a chemically synthesized peptide. The extracellular matrix molecule may have a repeat of the Gly-X-Y sequence characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. Multiple Gly-X-Y residues may be the same or different. The repeat of the Gly-X-Y sequence reduces constraints on the molecular chain configuration. In an extracellular matrix molecule having a repeat of the Gly-X-Y sequence, the proportion of the Gly-X-Y sequence of the total amino acid sequence may be 80% or more, preferably 95% or more. The extracellular matrix molecule may be a polypeptide having an RGD sequence. The RGD sequence is a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). Extracellular matrix molecules containing a sequence represented by Gly-X-Y and an RGD sequence include collagen, fibronectin, vitronectin, laminin, cadherin, etc.

Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specific examples include type I collagen, type II collagen, and type III collagen. Examples of non-fibrous collagen include type IV collagen. The collagen is preferably fibrous collagen.

Proteoglycans include, but are not limited to, chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.

The extracellular matrix component may contain at least one selected from the group consisting of collagen, laminin, and fibronectin, and preferably contains collagen from the viewpoint of excellent cell adhesiveness. The collagen is preferably fibrous collagen, more preferably type I collagen. Commercially available collagen may be used as the fibrous collagen, and a specific example is type I collagen derived from porcine skin manufactured by Nippon Meat Packers, Ltd.

The extracellular matrix components may be derived from animals. Examples of animal species from which the extracellular matrix components are derived include, but are not limited to, humans, pigs, and cows. The extracellular matrix components may be derived from a single type of animal, or may be derived from a combination of multiple types of animals.

As used herein, "fragmentation" refers to breaking down aggregates of extracellular matrix molecules into smaller sizes. Fragmentation may be performed under conditions that sever the bonds within the extracellular matrix molecules, or under conditions that do not sever the bonds within the extracellular matrix molecules. Fragmented extracellular matrix components may include defibrated extracellular matrix components (defibrated extracellular matrix components), which are components obtained by defibrating the above-mentioned extracellular matrix components through the application of physical force. Defibration is one form of fragmentation, and is performed, for example, under conditions that do not sever the bonds within the extracellular matrix molecules.

The method for fragmenting extracellular matrix components is not particularly limited. Methods for defibrating extracellular matrix components include, for example, applying physical force using an ultrasonic homogenizer, agitator homogenizer, or high-pressure homogenizer. When using agitator homogenizer, the extracellular matrix components may be homogenized directly, or in an aqueous medium such as physiological saline. Furthermore, by adjusting the homogenization time, number of times, etc., it is possible to obtain millimeter- or nanometer-sized defibrated extracellular matrix components. Defibrated extracellular matrix components can also be obtained by defibrating the components through repeated freezing and thawing.

The fragmented extracellular matrix component may at least partially contain a defibrated extracellular matrix component. The fragmented extracellular matrix component may consist solely of a defibrated extracellular matrix component. That is, the fragmented extracellular matrix component may be a defibrated extracellular matrix component. The defibrated extracellular matrix component preferably contains a defibrated collagen component. The defibrated collagen component preferably maintains the triple helix structure derived from collagen. The defibrated collagen component may be a component that completely or partially maintains the triple helix structure derived from collagen.

Examples of the shape of the fragmented extracellular matrix components include fibrous shapes. "Fibrous" refers to a shape composed of thread-like fragmented extracellular matrix components, or a shape composed of thread-like fragmented extracellular matrix components cross-linked intermolecularly. At least a portion of the fragmented extracellular matrix components may be fibrous. Fibrous extracellular matrix components include thin thread-like objects (fibrils) formed by the aggregation of multiple thread-like extracellular matrix molecules, thread-like objects formed by the further aggregation of fibrils, and defibrillated versions of these thread-like objects. In fibrous extracellular matrix components, the RGD sequence is preserved and not destroyed.

The average length of the fragmented extracellular matrix components may be 100 nm or more and 400 μm or less, 100 nm or more and 200 μm or less, or 600 nm or more and 30 μm or less. In one embodiment, the average length of the fragmented extracellular matrix components may be 1 μm or more and 400 μm or less, 5 μm or more and 400 μm or less, 10 μm or more and 400 μm or less, 22 μm or more and 400 μm or less, or 100 μm or more and 400 μm or less. In other embodiments, 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, or 1 μm or less. In other embodiments, the average length of the fragmented extracellular matrix components may be 100 nm or more, 200 nm or more, 400 nm or more, 600 nm or more, 800 nm or more, 1 μm or more, 5 μm or more, 10 μm or more, 22 μm or more, or 100 μm or more. The average length of the majority of the fragmented extracellular matrix components may be within the above numerical range. Specifically, the average length of 95% of the fragmented extracellular matrix components may be within the above numerical range. The fragmented extracellular matrix components may be fragmented collagen components having an average length within the above range, or defibrated collagen components having an average length within the above range.

The average diameter of the fragmented extracellular matrix component may 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 defibrated collagen component having an average diameter within the above range.

The average length and average diameter of fragmented extracellular matrix components can be determined by measuring individual fragmented extracellular matrix components using an optical microscope and analyzing the images. In this specification, "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.

The fragmented extracellular matrix component may, for example, contain a fragmented collagen component or may consist of a fragmented collagen component. "Fragmented collagen component" refers to a collagen component, such as a fibrous collagen component, that has been fragmented and 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 40 μm, more preferably 4 μm to 40 μm, and even more preferably 10 μm to 30 μm.

The concentration of fragmented extracellular matrix components can be determined appropriately depending on the shape and thickness of the desired three-dimensional tissue, the size of the culture vessel, etc.

The amount of fragmented extracellular matrix components may be, for ^example , 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 per 1.0 x 10 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.

The mass ratio of fragmented extracellular matrix components to cells (fragmented extracellular matrix components/cells) may be 1/1 to 1000/1, 9/1 to 900/1, or 10/1 to 500/1.

The content of fragmented extracellular matrix components in the mixture may be, based on the total amount of the mixture, 0.1 w/v% or more, 0.2 w/v% or more, 0.3 w/v% or more, 0.4 w/v% or more, 0.5 w/v% or more, 0.6 w/v% or more, 0.7 w/v% or more, 0.8 w/v% or more, 0.9 w/v% or more, 1.0 w/v% or more, or 1.1 w/v% or more, or 90 w/v% or less, 60 w/v% or less, 30 w/v% or less, 10 w/v% or less, 5.0 w/v% or less, 3.0 w/v% or less, 2.0 w/v% or less, 1.5 w/v% or less, 1.2 w/v% or less, 0.9 w/v% or less, 0.8 w/v% or less, 0.7 w/v% or less, or 0.6 w/v% or less. Since this makes it easier to suppress variation in the structure of the three-dimensional mammary gland model that is formed, the content of the fragmented extracellular matrix component in the mixture may be, based on the total amount of the mixture, 0.1 w/v% to 0.8 w/v%, 0.1 w/v% to 0.7 w/v%, 0.1 w/v% to 0.6 w/v%, 0.2 w/v% to 0.8 w/v%, 0.2 w/v% to 0.7 w/v%, 0.2 w/v% to 0.6 w/v%, 0.3 w/v% to 0.8 w/v%, 0.2 w/v% to 0.7 w/v%, or 0.3 w/v% to 0.6 w/v%.

(Alginate gel precursor)
The mixture contains an alginate gel precursor, which is a substance that reacts with a gelation accelerator to produce an alginate gel. The alginate gel has a three-dimensional network structure formed by cross-linking multiple carboxyl groups in alginate molecules.

By performing differentiation-inducing culture in an alginate gel formed using an alginate gel precursor, adipose-derived stem cells are more likely to differentiate into mature adipocytes. Therefore, by using an alginate gel precursor, it is possible to produce a three-dimensional mammary gland model without using mature adipocytes extracted from the body's adipose tissue.

The gelation accelerator for forming an alginate gel may be a polyvalent cation. That is, the alginate gel may have a structure in which carboxyl groups in alginate molecules are crosslinked by polyvalent cations. Examples of polyvalent cations include calcium ions (Ca ²⁺ ), barium ions (Ba ²⁺ ), magnesium ions (Mg ²⁺ ), iron(III) ions (Fe ³⁺ ), and aluminum ions (Al ³⁺ ).

The content of the alginate gel precursor may be 0.05 w/v% or more, 0.10 w/v% or more, 0.15 w/v% or more, 0.20 w/v% or more, or 0.25 w/v% or more, based on the total amount of the mixture. Since this makes it easier to obtain a three-dimensional mammary gland model, the content of the alginate gel precursor may be 3.0 w/v% or less, 1.0 w/v% or less, 0.80 w/v% or less, 0.50 w/v% or less, or 0.30 w/v% or less, based on the total amount of the mixture. From the viewpoint of facilitating the formation of acinar structures, the content of the alginate gel precursor may be, for example, 0.20 w/v% or more and less than 1.0 w/v%, 0.20 w/v% or more and 0.80 w/v% or less, 0.20 w/v% or more and 0.50 w/v% or less, or 0.20 w/v% or more and 0.30 w/v% or less, based on the total amount of the mixture.

(Mixing method)
The mixing step may be carried out by mixing aqueous media containing the adipose-derived stem cells and mammary epithelial cells, the fragmented extracellular matrix components, and the alginate gel precursor in any order.

"Aqueous medium" refers to a liquid containing water as an essential component. Specific examples of aqueous media include, but are not limited to, water such as ultrapure water, physiological saline such as phosphate-buffered saline (PBS), and liquid media such as Dulbecco's Modified Eagle Medium (DMEM) and medium specifically for mammary epithelial cells (MEGM). The liquid medium may also be a mixed medium made by mixing two or more types of media.

The mixing step may include stirring, incubating for a certain period of time, etc., as necessary after mixing each component and/or after mixing all components.

The mixing step may be carried out, for example, by mixing a liquid containing fragmented extracellular matrix components and an aqueous medium with a liquid containing cells and an aqueous medium, and then mixing the resulting mixture with a liquid containing an alginate gel precursor and an aqueous medium. The aqueous media contained in the liquids containing each component may be the same or different.

<Gelling step>
In the gelation step, the mixture containing the cells, the fragmented extracellular matrix components, and the alginate gel precursor is allowed to gel.

Gelling can be achieved by reacting the alginate gel precursor in the mixture with polyvalent cations. Gelation can be achieved, for example, by contacting the mixture with polyvalent cations in an aqueous medium. Examples of polyvalent cation sources, which are materials that provide polyvalent cations in an aqueous medium, include calcium salts (e.g., calcium chloride), barium salts, magnesium salts, iron salts, and aluminum salts.

Gelling may be a process in which cells and fragmented extracellular matrix components are embedded in an alginate gel.

The concentration of the polyvalent cations may be 50 mM, 60 mM or more, 80 mM or more, or 90 mM or more, and may be 150 mM or less, 120 mM or less, or 110 mM or less, based on the total volume of the solution containing the polyvalent cations. The concentration of the polyvalent cations may be 50 mM or more and 150 mM or less, 120 mM or less, or 110 mM or less, based on the total volume of the solution containing the polyvalent cations, 60 mM or more and 150 mM or less, 120 mM or less, or 110 mM or less, 80 mM or more and 150 mM or less, 120 mM or less, or 110 mM or less, and 90 mM or more and 150 mM or less, 120 mM or less, or 110 mM or less.

The gelation step may include incubating for a certain period of time to allow gelation to occur. The incubation temperature for gelation may be 20°C to 40°C, or 30°C to 37°C. The incubation time for gelation may be, for example, 5 minutes or more, or 10 minutes or more, and 60 minutes or less, or 30 minutes or less. The incubation time for gelation may be 5 minutes or more and 60 minutes or less, or 30 minutes or less, or 10 minutes or more and 60 minutes or less, or 30 minutes or less.

The gelation step may include washing the gelled mixture, if necessary. Washing can be performed using an aqueous medium (e.g., PBS) or the like.

<Culture process>
In the culture step, the gelled mixture is subjected to differentiation-inducing culture, whereby at least a portion of the adipose-derived stem cells are differentiated into mature adipocytes to form a three-dimensional tissue. As used herein, "differentiation-inducing culture of the gelled mixture" refers to culturing the gelled mixture under conditions that promote differentiation of the adipose-derived stem cells in the gelled mixture into mature adipocytes. The differentiation-inducing culture is not particularly limited as long as it promotes differentiation of the adipose-derived stem cells into mature adipocytes. For example, from the perspective of more efficiently differentiating the adipose-derived stem cells into mature adipocytes, the gelled mixture may be cultured in a medium containing fatty acids. The culture step can be performed, for example, by adding the gelled mixture to a medium containing fatty acids after washing as necessary, and culturing at a predetermined temperature for a predetermined period of time. If necessary, preliminary culture may be performed prior to the differentiation-inducing culture, in which the gelled mixture is cultured in a medium.

Culture media used in the culture process include, for example, mammary epithelial cell culture media (e.g., MEGM medium (manufactured by Lonza)), KBM medium, etc. The medium may be a serum-supplemented medium or a serum-free medium. The medium may also be a medium supplemented with growth factors, hormones such as insulin, etc. The medium may also be a mixed medium made by mixing two types of medium.

When the medium contains fatty acids, the content of the fatty acids in the medium may be, for example, 0.1 μM to 200 μM, 2 μM to 100 μM, 10 μM to 60 μM, 30 μM to 50 μM, 30 μM to 150 μM, or 80 μM to 120 μM; or 1 μM or more, 5 μM or more, 10 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, 50 μM or more, 60 μM or more, 70 μM or more, 80 μM or more, 90 μM or more; or 200 μM or less, 150 μM or less, 120 μM or less, 100 μM or less, 80 μM or less, or 70 μM or less. When the medium contains two or more fatty acids, the content of the fatty acids in the medium mentioned above refers to the content of each fatty acid contained in the medium.

When the medium contains fatty acids, the amount of fatty acids may be, for example, 2.0×10 ⁻¹¹ mol to 4.0×10 ⁻⁸ mol, or 1.0×10 ⁻⁹ mol to 2.0×10 ⁻⁸ mol per 1×10 ⁶ cells of adipose-derived stem cells. When the medium contains fatty acids, the weight of each fatty acid may be, for example, 6 ng to 15 μg, or 250 ng to 10 μg per 1×10 ⁶ cells of adipose-derived stem cells.

The culture medium may contain a substance that stimulates milk secretion. Examples of substances that stimulate milk secretion include prolactin and oxytocin.

The cell density in the medium in the culturing step can be appropriately determined depending on the shape and thickness of the desired three-dimensional tissue, the size of the culture vessel, etc. For example, the cell density in the medium in the culturing step may be 1 to ¹⁰ cells/mL, or ¹⁰ to ¹⁰ cells/mL. Furthermore, the cell density in the medium in the culturing step may be the same as the cell density in the aqueous medium in the mixing step.

There are no particular restrictions on the culture conditions, and suitable conditions can be set appropriately depending on the type of cells being cultured, etc. 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 6.0 to 8.0, or 7.2 to 7.4. The culture time may be 24 hours to 336 hours, 72 hours to 336 hours, 96 hours to 384 hours, or 96 hours to 288 hours.

The culture vessel (support) used for cell culture 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 detached from the support during the culture process. When culturing the cells without being attached to the support, or when culturing the cells after being detached from the support during the culture process, 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. When culturing the cells without being attached to the support, ball-shaped three-dimensional tissues are likely to form.

The culturing step may include embedding the gelled mixture in Matrigel. The embedding of the gelled mixture in Matrigel may be performed before starting culturing the gelled mixture in a medium.

Matrigel is a protein mixture containing proteins that make up basement membranes. The main components of Matrigel are laminin, type IV collagen, entactin, and heparan sulfate proteoglycan. Matrigel can be obtained by extracting proteins that make up basement membranes from Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Commercially available Matrigel products include Corning® Matrigel Basement Membrane Matrix (manufactured by Corning).

Specific methods for embedding the gelled mixture in Matrigel include filling a culture vessel with Matrigel and leaving the gelled mixture therein, and leaving the gelled mixture in a culture vessel and then filling it with Matrigel.

The culturing step may be carried out in the presence of fibroblasts. That is, the culture medium may contain fibroblasts. The fibroblasts may be human mammary fibroblasts (HMF). When a three-dimensional tissue construct is produced by embedding the gelled mixture in Matrigel and then culturing it in the presence of fibroblasts, the acinar structure of the three-dimensional construct becomes more stable, making it easier to obtain a more developed acinar structure.

Culturing of the gelled mixture in the presence of fibroblasts can be carried out using a container equipped with a substrate (permeable membrane) that allows liquid to pass through but does not allow cells in the liquid to pass through. Examples of containers equipped with permeable membranes include, but are not limited to, cell culture inserts such as Transwell® inserts, Netwell® inserts, Falcon® cell culture inserts, and Millicell® cell culture inserts.

The gelled mixture can be cultured in the presence of fibroblasts by the following method. Figure 17 is a schematic diagram illustrating a method for culturing a gelled mixture in the presence of fibroblasts. First, the gelled mixture 1 and Matrigel 4 in which the mixture is embedded are placed on a permeable membrane 10a, and fibroblasts 3 are placed on the bottom of the container 10. Culture medium 2 is then placed in the container 10, and the gelled mixture 1 and fibroblasts 3 embedded in Matrigel 4 are cultured.

The method according to this embodiment may include, after the culturing step, a step of further culturing the three-dimensional tissue in a medium containing fatty acids. When this step is included, the formation of acinar structures and the synthesis of milk components are improved. The culturing of the three-dimensional tissue in a medium containing fatty acids can be carried out, for example, under the same culture conditions as those described above.

The method according to this embodiment makes it possible to produce a three-dimensional mammary gland model without using mature adipocytes collected from the adipose tissue of a living body.

The method according to this embodiment makes it possible to produce a three-dimensional mammary gland model with a structure closer to that of a living body. For example, the method according to this embodiment makes it possible to obtain a three-dimensional mammary gland model with an alveolar structure.

Acinar structure refers to a structure similar to the acini present at the end of an exocrine gland. The acinar structure of the three-dimensional mammary gland model is formed by the aggregation of multiple mature adipocytes. The acinar structure of the three-dimensional mammary gland model may further contain mammary epithelial cells. The acinar structure of the three-dimensional mammary gland model can be said to be a structure formed by the aggregation of two or more cells selected from the group consisting of mature adipocytes and mammary epithelial cells.

The acinar structure may be composed of a lobe and a stalk. The lobe of the acinar structure contains mature adipocytes and is a region where aggregates containing multiple mature adipocytes are present. The aggregates in the lobe of the acinar structure may further contain mammary epithelial cells. The aggregates containing multiple mature adipocytes may be surrounded by fragmented extracellular matrix components (e.g., fragmented collagen components).

In a three-dimensional mammary gland model consisting of a three-dimensional tissue containing mammary epithelial cells and mature adipocytes, if there is an area where multiple mature adipocytes aggregate, it can be determined that the model has an acinar structure. In other words, if the three-dimensional mammary gland model according to this embodiment contains two or more aggregates formed by the aggregation of two or more mature adipocytes, where the lipid droplets have a diameter of 20 μm or more, it can be said to contain an acinar structure. The presence of mammary epithelial cells can be confirmed by CK8/18 staining. The presence of mature adipocytes can be confirmed by staining with Nile Red, a marker for lipid droplets and adipocytes.

The method according to this embodiment makes it possible to obtain a three-dimensional mammary gland model in which, for example, two mammary epithelial cell layers exist. This type of two-layer structure is also found in living mammary glands, making this model more suitable as a mammary gland model. The existence of two mammary epithelial cell layers can be confirmed by a method using marker proteins. When a layer of CK8-positive cells and a layer of CK14-positive cells exist, it can be determined that two mammary epithelial cell layers exist. As used herein, "positive cells" refers to cells that express a specific marker protein on or within the cell surface.

The method according to this embodiment is not limited to those comprising the mixing step, gelling step, and culturing step described above. For example, in the culturing step, a medium without added fatty acids (a medium without fatty acids supplied from an external source) can be used instead of a medium containing fatty acids. That is, the medium used in the culturing step may or may not contain fatty acids. When a medium without added fatty acids is used, the three-dimensional mammary gland model may be cultured in a medium containing fatty acids after it has been produced. Specifically, after the step of differentiating at least a portion of the adipose-derived stem cells into mature adipocytes to form a three-dimensional tissue, the three-dimensional tissue may be further cultured in a medium containing fatty acids. Fatty acids may be added to the medium at any timing during the culturing step, such as when milk components are produced.

[Three-dimensional mammary gland model]
The three-dimensional mammary gland model according to this embodiment comprises a three-dimensional tissue structure including cells, including mature adipocytes and mammary epithelial cells, fragmented extracellular matrix components, and alginate gel. The three-dimensional mammary gland model can be suitably produced by the method described above. The three-dimensional mammary gland model may have the above-described alveolar structure. The three-dimensional mammary gland model may have the above-described two-layer mammary epithelial cell layer.

A three-dimensional mammary gland model is a mammary gland model that can be artificially formed in vitro, and does not include mammary gland tissue itself that has simply been isolated from a living organism. However, the structure of living organism's mammary gland tissue is more complex than that of three-dimensional tissue. Therefore, the three-dimensional mammary gland model can be easily distinguished from three-dimensional tissue and living organism's mammary gland tissue.

As used herein, the term "three-dimensional tissue" refers to an aggregate of cells (agglomerated cell mass) in which cells are arranged three-dimensionally via extracellular matrix components and/or fragmented extracellular matrix components, and refers to an aggregate artificially created by cell culture. The shape of the three-dimensional tissue 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. From the perspective of being closer to the mammary gland in vivo, the shape of the three-dimensional tissue is preferably spherical or approximately spherical. The diameter of a spherical or approximately spherical three-dimensional tissue may be 0.5 to 3 mm.

The total number of cells that make up the three-dimensional tissue is not particularly limited and is determined appropriately taking into consideration the thickness and shape of the three-dimensional mammary gland model to be constructed, the size of the cell culture vessel used for construction, etc. The total number of cells that make up the three-dimensional tissue is also synonymous with the total number of cells that make up the three-dimensional mammary gland model.

In the three-dimensional tissue according to this embodiment, the cells contain at least mature adipocytes and mammary epithelial cells. Because the three-dimensional tissue according to this embodiment is used as a mammary gland model, it is preferable to use mature adipocytes derived from mammary gland adipose tissue.

The content of mature adipocytes 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 three-dimensional tissue, or 95% or less, 90% or less, 80% or less, or 75% or less. From the perspective of being more suitable as a mammary gland model, the content of mature adipocytes may be 30% or more and 80% or less, 35% or more and 75% or less, or 40% or more and 65% or less of the total number of cells in the three-dimensional tissue.

The content of mammary epithelial cells relative to the total number of cells in the three-dimensional tissue may 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, or may 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. From the perspective of being more suitable as a mammary gland model, the content of mammary epithelial cells relative to the total number of cells in the three-dimensional tissue may be 15% or more and 70% or less, 15% or more and 35% or less, or 20% or more and 30% or less.

In the three-dimensional tissue according to this embodiment, the ratio (cell number) of mature adipocytes to mammary epithelial cells is not particularly limited, but may be, for example, 0.25:1 to 2.5:1, or 1:1 to 2.4:1. In the three-dimensional tissue according to this embodiment, the ratio of the number of mature adipocytes to the number of mammary epithelial cells may be within the numerical range described above as the ratio of the number of adipose-derived stem cells to the number of mammary epithelial cells.

The cells in the three-dimensional tissue may contain undifferentiated fat cells such as adipose-derived stem cells. The content of adipose-derived stem cells may be, for example, 1% or more, 3% or more, 5% or more, 10% or more, or 15% or more of the total number of cells in the three-dimensional tissue, or 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less. 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 and 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less; 3% or more and 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less; 5% or more and 40% or less, 30% or less, 20% or less, or 10% or less; 10% or more and 40% or less, 30% or less, or 20% or less; or 15% or more and 40% or less, 30% or less, or 20% or less.

The content of extracellular matrix components in the three-dimensional tissue may be 0.01 to 90% by mass, preferably 10 to 90% by mass, preferably 10 to 80% by mass, preferably 10 to 70% by mass, preferably 10 to 60% by mass, preferably 1 to 50% by mass, preferably 10 to 50% by mass, more preferably 10 to 30% by mass, and more preferably 20 to 30% by mass, based on the three-dimensional tissue (dry weight).

Here, "extracellular matrix components in a three-dimensional tissue" refers to the extracellular matrix components that make up the three-dimensional tissue, and may be derived from endogenous extracellular matrix components or exogenous extracellular matrix components.

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

"Exogenous extracellular matrix components" refers to extracellular matrix components supplied from the outside. The three-dimensional tissue of 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.

In other words, when a three-dimensional tissue contains endogenous extracellular matrix components and fragmented extracellular matrix components, the content of extracellular matrix components constituting the three-dimensional tissue refers to the total amount of endogenous extracellular matrix components and fragmented extracellular matrix components. The extracellular matrix content can be calculated from the volume of the obtained three-dimensional tissue and the mass of the decellularized three-dimensional tissue.

When the extracellular matrix component is a collagen component, the exogenous extracellular matrix component is also referred to as an "exogenous collagen component." An "exogenous collagen component," meaning a collagen component supplied from the outside, is an aggregate of collagen molecules formed by multiple collagen molecules, and specific examples include fibrous collagen and non-fibrous collagen. The exogenous collagen component is preferably fibrous collagen. The above-mentioned fibrous collagen refers to the collagen component that is the main component of collagen fibers, and examples include type I collagen, type II collagen, and type III collagen. The above-mentioned fibrous collagen may be commercially available collagen, and a specific example is type I collagen derived from porcine skin manufactured by Nippon Meat Packers, Ltd. An example of exogenous non-fibrous collagen is type IV collagen.

In exogenous extracellular matrix components, the animal species from which they are derived may be different from that of the cells. Also, if the cells include extracellular matrix-producing cells, the animal species from which they are derived may be different from that of the extracellular matrix-producing cells. In other words, the exogenous extracellular matrix components may be heterologous extracellular matrix components.

For example, when the extracellular matrix component contained in a three-dimensional tissue is a collagen component, the amount of collagen in the three-dimensional tissue can be quantified by, for example, quantifying hydroxyproline as follows: A solution containing the three-dimensional tissue is mixed with hydrochloric acid (HCl), incubated at high temperature for a predetermined time, then returned to room temperature. The supernatant is centrifuged and 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 specified with a hydroxyproline assay buffer and a detection reagent, and the absorbance at 570 nm is measured. The amount of collagen is calculated by comparing the absorbance of the sample with the standard. Alternatively, the three-dimensional tissue may be directly suspended in high-concentration hydrochloric acid, the resulting solution is centrifuged, and the supernatant is recovered and used to quantify the collagen component. The three-dimensional tissue to be dissolved may be in the state it was recovered from the culture medium, or it may be dried after recovery to remove the liquid components. However, when quantifying collagen components by dissolving a three-dimensional tissue directly after recovery from the culture medium, it is expected that the measured value of the three-dimensional tissue weight will vary due to the influence of medium components absorbed by the three-dimensional tissue and residual medium due to experimental techniques. Therefore, from the perspective 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.

Specific examples of methods for quantifying the amount of collagen components include the following:

(Sample Preparation)
The entire volume of the freeze-dried three-dimensional tissue was mixed with 6 mol/L HCl, incubated at 95°C in a heat block for at least 20 hours, and then returned to room temperature. After centrifugation at 13,000 g for 10 minutes, the supernatant of the sample solution was collected. After diluting appropriately with 6 mol/L HCl so that the results in the measurement described below fall within the range of the calibration curve, 200 μL was diluted with 100 μL of ultrapure water to prepare the sample. 35 μL of sample was used.

(Preparation of standards)
Add 125 μL of standard solution (1200 μg/mL in acetic acid) and 125 μL of 12 mol/L HCl to a screw-cap tube, mix, and incubate at 95°C in a heat block for 20 hours, then return to room temperature. After centrifugation at 13,000 g for 10 minutes, the supernatant was diluted with ultrapure water to prepare S1 at 300 μg/mL. S1 was then serially diluted 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). S8 (0 μg/mL) containing 90 μL of 4 mol/L HCl alone was also prepared.

Assay
Add 35 μL of the standard and sample to each 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. Peel off 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 absorbance of the sample with that of the standard.

The collagen component in the three-dimensional tissue may be defined by its area ratio or volume ratio. "Defining by area ratio or volume ratio" means, for example, making the collagen component in the three-dimensional tissue 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 three-dimensional tissue using naked eye observation, various microscopes, image analysis software, etc. When defining by area ratio, there are no limitations on which cross section or surface in the three-dimensional tissue is used to define the area ratio; for example, if the three-dimensional tissue is a sphere, it may be defined by a cross section passing through its approximate center.

For example, when defining the collagen component in a three-dimensional tissue by area ratio, the area ratio is 0.01 to 99% of the total area of the three-dimensional tissue, preferably 1 to 99%, preferably 5 to 90%, preferably 7 to 90%, preferably 20 to 90%, and more preferably 50 to 90%. The "collagen component in a three-dimensional tissue" is as described above. The area ratio of the collagen component constituting the three-dimensional tissue refers to the combined area ratio of endogenous collagen components and exogenous collagen components. The area ratio 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 blue-stained collagen component to the total area of a cross section passing through approximately the center of the three-dimensional tissue.

The three-dimensional tissue contains alginate gel. The three-dimensional tissue is preferably embedded in alginate gel. "The three-dimensional tissue is embedded in alginate gel" means that alginate gel is present on the outside of the three-dimensional tissue, or in at least some or all of the intercellular spaces on the outside and inside of the three-dimensional tissue. The three-dimensional tissue may contain cells and fragmented extracellular matrix components, and alginate gel encapsulating these.

The three-dimensional structure may further contain a fatty acid. The number of carbon atoms in the fatty acid may be 10 or more, 12 or more, 14 or more, 16 or more, or 17 or more, and may be 25 or less, 22 or less, 20 or less, or 19 or less, or may be 18. The fatty acid may be a saturated fatty acid or an unsaturated fatty acid. If the fatty acid is unsaturated, the number of carbon-carbon double bonds in the molecule may be 1 or more, 3 or less, or 2 or less, or may be 1. Examples of fatty acids include oleic acid, erucic acid, elaidic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid. One type of fatty acid may be used alone, or two or more types may be used in combination. The fatty acid may be derived from a component in the culture medium during the production process.

The thickness of the three-dimensional tissue may 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 three-dimensional tissues have a structure closer to that of living tissue, making them suitable as substitutes for laboratory animals and transplant materials. There is no particular upper limit to the thickness of the three-dimensional tissue, but it may be, for example, 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.

Here, "thickness of a three-dimensional structure" means the distance between both ends in the direction perpendicular to the main surface when the three-dimensional structure is rectangular. When the main surface has irregularities, the thickness means the distance at the thinnest part of the main surface.

When the three-dimensional structure is spherical or approximately spherical, the thickness of the three-dimensional structure refers to the diameter of the three-dimensional structure. When the three-dimensional structure is ellipsoidal or approximately ellipsoidal, the thickness of the three-dimensional structure refers to the minor axis of the three-dimensional structure. When the three-dimensional structure is approximately spherical or approximately ellipsoidal and has an uneven surface, the thickness of the three-dimensional structure refers to the shortest distance between the two points where a line passing through the center of gravity of the three-dimensional structure intersects with the surface.

The three-dimensional tissue according to this embodiment is constructed in a cell culture vessel. The cell culture vessel is not particularly limited, as long as it is capable of constructing a three-dimensional tissue and culturing the constructed three-dimensional tissue. Specific examples of the cell culture vessel include dishes, cell culture inserts (e.g., Transwell® inserts, Netwell® inserts, Falcon® cell culture inserts, Millicell® cell culture inserts, etc.), tubes, flasks, bottles, plates, etc. When constructing a three-dimensional tissue, dishes or various cell culture inserts are preferred, as this allows for more accurate evaluation using the three-dimensional tissue. The three-dimensional tissue may be constructed by non-adherent culturing of cells in the cell culture vessel. By non-adherent culturing of cells in the cell culture vessel, it is easy to construct ball-shaped three-dimensional tissues.

The following provides a more detailed explanation based on examples. However, this embodiment is not limited to the following examples.

Mammary epithelial cells (HMEC) used were Lonza Co., Ltd. model number CC-2551.

The adipose-derived human stem cells used were Lonza Co., Ltd. model number PT-5006. These cells are adipose-derived mesenchymal stem cells (hADSCs).

<Preparation of alginate solution>
1 mg of sodium alginate (Fujifilm Wako Pure Chemical Industries, Ltd., model number 9005-38-3) was dissolved in 100 μL of PBS and incubated for 5 minutes at 37° C. The resulting liquid was filtered through a 0.22 μm filter to obtain an alginic acid solution.

<Preparation of _CaCl2 solution>
11.1 mg of calcium chloride was dissolved in 1 mL of PBS to obtain a _CaCl2 solution.

<Preparation of BSA-oleic acid>
176 mg of sodium chloride was dissolved in 20 ml of Milli-Q to obtain a 150 mM NaCl solution. 1.06 g of bovine serum albumin (BSA) was added to the 150 mM sodium chloride solution and vortexed for several minutes to obtain a 0.8 mM BSA solution. 31.5 μL of oleic acid was added to the BSA solution and vortexed for several minutes to obtain a BSA-oleic acid solution. The pH of the BSA-oleic acid solution was adjusted to 7.4 using NaOH. The pH-adjusted BSA-oleic acid solution was warmed in a 37°C water bath for 2 hours. After warming, the BSA-oleic acid solution was filtered through a 0.22 μm filter and used for testing.

<Preparation of Fragmented Collagen Component (CMF)>
A neutralization buffer consisting of 2.125 mL of 0.05 N NaOH and 2.125 mL of 10x PBS was added to 17 mL of 3 mg/mL collagen solution and heated at 37°C for 30 minutes to allow gelation. The resulting gel was freeze-dried for 72 hours to obtain freeze-dried collagen after gelation. 15 mL of 85 v/v% ethanol was added to the gelated freeze-dried collagen and defibrated for 6 minutes using a homogenizer (probe: S10N-10G-ST shaft generator). The samples obtained after defibration were centrifuged at 10,000 rpm, the supernatant was discarded, and 15 mL of 70 v/v% ethanol was added to each sample. The samples were then pipetted using a homogenizer (S10N-8G shaft generator). The samples obtained after pipetting were centrifuged at 10,000 rpm, the supernatant was discarded, and 15 mL of ultrapure water was added and pipetted. The samples were then sonicated 10 times for 20 seconds using an ultrasonic homogenizer. The sample containing defibrated collagen (CMF) obtained by the above procedure was freeze-dried while the CMF remained dispersed in water. This resulted in fragmented collagen components (CMF) with diameters of approximately 20-30 μm and lengths of approximately 100-200 μm. The diameter and length of the CMF were determined by analyzing each fragmented collagen component using an electron microscope.

<Creation of three-dimensional tissue>
The pre-cultured ADSCs and HMECs were harvested and counted. The required number of cells and materials was calculated. The required amount of CMF was measured and vortexed with 1 ml of Milli-Q. The vortexed CMF-containing solution was centrifuged at 10,000 rpm for 3 minutes, the supernatant was discarded, and 500 μL of Milli-Q was added and vortexed. After vortexing, 100 μL of the CMF-containing solution was dispensed and mixed with the required amount of cell suspension. The mixture was then centrifuged at 9,000 g for 1 minute and the supernatant was removed. PBS was added to each tissue to make a total volume of 3.75 μL. Then, an alginate solution was added to each tissue to achieve the desired alginate content in the mixture, and the mixture was mixed. This method yielded a mixture containing alginate, CMF, and cells.

The ratio of ADSC cell number to HMEC cell number was 1. The cell concentration in the mixture was 6 x ¹⁰ cells/mL. The alginate content in the mixture was 0.25 w/v% based on the total mixture volume. The CMF content in the mixture was 0 w/v%, 0.3 w/v%, 0.6 w/v%, 0.9 w/v%, or 1.2 w/v% based on the total mixture volume.

100 μL of 100 mM CaCl ₂ solution was dispensed into a non-adhesive 96-well plate, and 5 μL of the mixture prepared as above was added dropwise to the plate to gel the mixture.

The gelled mixture was incubated at 37°C for 15 minutes, then scooped up with a spatula and washed three times with PBS. The gelled mixture was cultured in 300 μL of MEGM medium for two days, then transferred to a 24-well non-adhesive plate containing 500 μL of MEGM containing 750 mM BSA-oleic acid and 1 μg/ml prolactin.

The gelled mixture was transferred to a 24-well non-adhesive plate and cultured for the specified period (7 or 14 days) with medium changes every 2-3 days. The resulting 3D tissues were fixed in a PBS solution of 1% PFA and 50 mM _CaCl2 at room temperature for 1 hour and stained with Nile red or CK8/18.

Figure 1 shows micrographs of the three-dimensional tissue. (A) shows the results of observation of the three-dimensional tissue on day 7 of culture, and (B) shows the results of observation of the three-dimensional tissue on day 14 of culture.

Figure 2 shows the results of Nile Red staining and DAPI staining of three-dimensional tissues cultured for 14 days. Figure 2(A) shows the results when the CMF content was 0%, and Figure 2(B) shows the results when the CMF content was 1.2%.

Figure 3 shows the results of Nile Red staining, DAPI staining, and CK8/18 staining of a three-dimensional tissue after a 14-day culture period. The three-dimensional tissue shown in Figure 3 was prepared under conditions where the CMF content was 0.6%.

Figure 4 shows the results of Nile Red staining and DAPI staining of a three-dimensional tissue cultured for seven days. The three-dimensional tissue shown in Figure 4(A) was prepared under conditions where the CMF content was 0.6 w/v%. The three-dimensional tissue shown in Figure 4(B) was prepared using non-fragmented collagen (Collagen Type I (Nippi, model number: 892171)) instead of CMF.

Three-dimensional tissues using dedifferentiated fat cells (DFAT) were prepared using the following method. Pre-cultured DFAT and HMEC were harvested and counted. The ratio of ADSC to HMEC cell count was set to 1. The cell concentration in the mixture was 6 x 10 ⁶ cells/mL. The necessary cells were collected, centrifuged, and the supernatant was removed. The fragmented collagen component was mixed to a final concentration of 0.6 v/v%. PBS was added to each tissue to a volume of 3.75 μL. Then, an alginate solution was added to each tissue to achieve the desired alginate content in the mixture and mixed. Using the above method, a mixture containing alginate, fragmented collagen component, DFAT, and HMEC was obtained. A three-dimensional tissue using DFAT was prepared using the same method as for preparing three-dimensional tissues using ADSCs, except for using the resulting mixture. DFAT was prepared in the same manner as described in paragraphs [0045] to [0058] of JP-A No. 2024-136632.

Figures 7-9 show the results of immunostaining for CK8 and CK14, α-lactalbumin (LALBA), butyrophilin (BTN1A1), and MFGE8 milk proteins in frozen sections of three-dimensional tissues made using ADSCs or DFAT. Figure 7 shows that immunostaining for CK8 and CK14 revealed the presence of two mammary epithelial cell layers in the three-dimensional tissues, whether ADSCs or DFAT cells were used. These results demonstrate that the three-dimensional tissues obtained using the above method contain differentiated mammary epithelial cells and are closer to the mammary gland in vivo.

Furthermore, as shown in Figures 8 and 9, the expression of BTN1A1 and MFGE8 proteins was similar whether ADSC or DFAT cells were used.

Figure 10 shows the results of quantification of human α-lactalbumin levels using an ELISA assay. DFAT cells produced 42% more α-lactalbumin than ADSC cells. α-lactalbumin (LALBA) accounts for approximately 22% of breast milk proteins and is thought to play an important role in lactose synthesis, which is essential for breast milk production (Non-Patent Document 3: Ogg, S. L., et al. 2004, PNAS, 101, 10084). Butyrophilin (BTN1A1) is a membrane protein associated with milk fat globules and plays an important role in lipid secretion (Non-Patent Document 4: Layman, D. K., et al. 2018, Nutrition Reviews, 76, 444).

Three-dimensional tissues using non-fragmented collagen were prepared using the following method. Pre-cultured ADSCs and HMECs were harvested and the cells were counted. The necessary cells were isolated and centrifuged, after which the supernatant was removed. Non-fragmented collagen was mixed to a final concentration of 0.6 v/v%. 3.75 μL of PBS was added per tissue. Then, alginate solution was added per tissue to achieve the desired alginate content in the mixture, and the mixture was mixed. Using the above method, a mixture containing alginate, non-fragmented collagen, and cells was obtained. A three-dimensional tissue using non-fragmented collagen was prepared using the same method as for preparing three-dimensional tissues using CMF, except that the resulting mixture was used.

Comparing Figures 4(A) and (B) shows that by using CMF, a three-dimensional mammary gland model with an acinar structure can be obtained.

Figures 11(A), (B), and (C) show the results of Nile Red staining and DAPI staining of three-dimensional tissues after a 14-day culture period. Figure 11(A) shows the results for a three-dimensional tissue prepared using CMF. Figure 11(B) shows the results for a three-dimensional tissue prepared without using either non-fragmented collagen or fragmented collagen components. Figure 11(C) shows the results for a three-dimensional tissue prepared using non-fragmented collagen instead of CMF.

The results shown in Figures 11(A), (B), and (C) indicate that a three-dimensional tissue was not formed in the absence of collagen, and that the three-dimensional tissue did not come together properly when non-fragmented collagen was used, suggesting that fragmented collagen may contribute to the formation of a three-dimensional tissue with an acinar structure. Figure 11(A) shows that the three-dimensional tissue created using CMF has an acinar structure.

Figures 5 and 6 show the results of bright-field observation of the three-dimensional tissue. Figures 5(A), (B), and (C) show the results when the CMF content was 0%, 0.3%, and 0.6%, respectively, and Figures 6(A) and (B) show the results when the CMF content was 0.9% and 1.2%, respectively.

As shown in Figures 5 and 6, it was confirmed that three-dimensional tissues could be obtained even when the CMF content was changed. Three-dimensional tissues with different CMF contents were produced multiple times, and it was confirmed that when the CMF content was 0.3% or 0.6%, three-dimensional tissue structures could be formed with less variation. Furthermore, when the content of alginate gel precursor (alginic acid) in the mixture was less than 1.0 w/v% based on the total amount of the mixture, acinar structures were more likely to form at all CMF contents compared to when this value was not met (1.0 w/v%).

A three-dimensional tissue using carrageenan was prepared using the following method. Pre-cultured ADSCs and HMECs were harvested and the cells were counted. The necessary cells were isolated and centrifuged, after which the supernatant was removed. Fragmented collagen was mixed to a final concentration of 0.6 v/v%. 3.75 μL of PBS was added per tissue. Carrageenan solution was then added per tissue to a carrageenan content of 0.25 v/v% in the mixture, and mixed. Using the above method, a mixture containing carrageenan, fragmented collagen components, and cells was obtained. A three-dimensional tissue using carrageenan was prepared using the same method as for preparing a three-dimensional tissue using alginate, except for using the resulting mixture.

Figure 12 shows the results of bright field observation of a three-dimensional tissue after a seven-day culture period. The three-dimensional tissue shown in Figure 12(A) was prepared using alginate. The three-dimensional tissue shown in Figure 12(B) was prepared using carrageenan (Sigma-Aldrich 22048) instead of alginate. These results show that mature adipocytes did not aggregate well with carrageenan, and no acinar structure was observed.

Three-dimensional tissues containing varying concentrations of oleic acid were prepared in the following manner: After gelation, a mixture of ADSCs (5 x ¹⁰ cells/mL) was washed three times with PBS and then transferred to a 24-well non-adhesive plate containing 500 μL of DMEM containing 500 μM or 750 μM BSA-oleic acid, instead of 500 μL of MEGM containing 750 mM BSA-oleic acid and 1 μg/mL prolactin. Three-dimensional tissues containing varying concentrations of oleic acid were prepared in the same manner as in the preparation of three-dimensional tissues using CMF, except that the mixture was transferred to a 24-well non-adhesive plate containing 500 μL of DMEM containing 500 μM or 750 μM BSA-oleic acid.

Figure 14 shows the results of Nile Red and DAPI staining of three-dimensional tissues after 7 days of culture. Figure 13 shows the fluorescence intensity ratio of Nile Red and DAPI (n=9). Figure 15 shows the number of lipid droplets with an area value of 50 ^μm2 or more counted in three-dimensional tissues after 7 days of culture, as measured by Image J (n=9). These results demonstrate that differences in oleic acid concentration do not affect the formation of three-dimensional tissues or lipid droplets in mature adipocytes.

Figure 16 shows the results of Nile Red and DAPI staining of three-dimensional tissues cultured for seven days when the ratio of ADSCs to HMECs was varied. It was found that a three-dimensional mammary gland model with an acinar structure was more easily obtained when the ratio of ADSCs to HMECs (ADSC/HMEC) was between 1/1 and 4/1.

The milk-synthesizing ability of three-dimensional tissues using CMF and alginate was investigated. After culturing for up to 7 days in a 24-well non-adhesive plate containing 500 μL of MEGM containing 750 mM BSA-oleic acid and 1 μg/ml prolactin (Fujifilm Wako Pure Chemical Industries, Ltd., prolactin, human, recombinant, product code: 166-29231), the tissues were harvested and washed with PBS. After placing them in assay buffer (contained in Lactose Assay Kit CBL, MET-5001) and pipetting, the three-dimensional structures were ultrasonically disrupted and centrifuged at 10,000 g for 10 minutes. The supernatant was then collected and lactose was detected using the Lactose Assay Kit (CBL, MET-5001).

Table 1 shows the average lactose concentration of three-dimensional tissues using CMF and alginate. It has been reported that human breast milk contains more than 50% lactose (Non-Patent Document 5: Sekerel BE. J Asthma Allergy. 2021 Sep 24;14:1147-1164.). Since lactose was detected, it was found that the mammary gland model of this embodiment synthesizes at least one component of milk. The SD in Table 1 indicates standard deviation.

Apart from the final culture step, three-dimensional tissues were created in the same way using CMF and alginate, except that the medium did not contain oleic acid. Specifically, the gelled mixture was incubated at 37°C for 15 minutes, then scooped up with a spatula and washed three times with PBS. The gelled mixture was embedded in Matrigel and cultured in a 24-well culture insert containing MEGM medium with HMF seeded on the bottom. The gelled mixture was cultured for the specified period (10 days), with the medium changed every 2-3 days.

Figure 18 shows the results of bright-field observation of a three-dimensional tissue after a 10-day culture period. The scale bar in Figure 18 indicates 500 μm. Figure 19 shows images of the three-dimensional tissue stained for nuclei (Nuclei) and F-actin, a merged image of these, and enlarged images of each image. Acinar structures were prominent in the medium containing HMF. Embedding the three-dimensional tissue in alginate gel and placing it in an HMF environment during the culture process may further stabilize the formation of acinar structures in the three-dimensional structure, leading to further development of the acinar structures. A more developed acinar structure is indicated by an increase in size in bright-field observation and the formation of acinar structures extending outward from the mammary tissue.

Claims (18)

A method for producing a three-dimensional mammary gland model consisting of a three-dimensional tissue structure containing mature adipocytes and mammary gland epithelial cells, comprising:
Gelling a mixture comprising cells, including adipose-derived stem cells and mammary epithelial cells, fragmented extracellular matrix components, and an alginate gel precursor; and
A method comprising a step of inducing differentiation of the gelled mixture to differentiate at least a portion of the adipose-derived stem cells into the mature adipocytes and form the three-dimensional tissue. The method of claim 1, wherein the fragmented extracellular matrix component comprises a fragmented collagen component. The method of claim 2, wherein the average length of the fragmented collagen component is 100 nm to 200 μm. The method of claim 2, wherein the fragmented collagen component is homogenized in an aqueous medium. The method of claim 1, wherein the differentiation-inducing culture is performed by culturing the gelled mixture in a medium containing fatty acids. The method of claim 5, wherein the fatty acid is oleic acid. The method of claim 1, further comprising, after the step of forming the three-dimensional tissue, a step of culturing the three-dimensional tissue in a medium containing a fatty acid. The method according to any one of claims 1 to 6, wherein the mixture is gelled by contacting it with calcium ions. The method according to any one of claims 1 to 6, wherein the content of the alginate gel precursor in the mixture is 0.20 w/v% or more and less than 1.0 w/v% based on the total amount of the mixture. The method according to any one of claims 1 to 6, wherein the content of fragmented extracellular matrix components in the mixture is 0.1 w/v% or more and 0.8 w/v% or less, based on the total amount of the mixture. A three-dimensional mammary gland model consisting of a three-dimensional tissue structure containing cells including mature adipocytes and mammary epithelial cells, fragmented extracellular matrix components, and alginate gel. The three-dimensional mammary gland model of claim 11, wherein the three-dimensional mammary gland model has an alveolar structure. The three-dimensional mammary gland model according to claim 11 or 12, wherein the fragmented extracellular matrix component contains a fragmented collagen component. The three-dimensional mammary gland model according to claim 11 or 12, wherein the three-dimensional tissue contains fatty acids. The three-dimensional mammary gland model of claim 14, wherein the fatty acid is oleic acid. The three-dimensional mammary gland model described in claim 11 or 12, constructed in a cell culture vessel. A method for producing a three-dimensional mammary gland model consisting of a three-dimensional tissue structure containing mature adipocytes and mammary gland epithelial cells, comprising:
Gelling a mixture containing cells including adipose-derived stem cells and mammary epithelial cells, a fragmented collagen component, and an alginate gel precursor; and
the step of culturing the gelled mixture in a medium containing oleic acid to differentiate at least a portion of the adipose-derived stem cells into the mature adipocytes and form acinar structures in the three-dimensional tissue;
the content of the alginate gel precursor in the mixture is 0.20 w/v% or more and 0.30 w/v% or less based on the total amount of the mixture,
The method, wherein the content of the fragmented collagen component is 0.1 w/v % or more and 0.8 w/v % or less based on the total amount of the mixture. A three-dimensional mammary gland model comprising a three-dimensional tissue containing cells including mature adipocytes and mammary epithelial cells, fragmented extracellular matrix components, and alginate gel, and containing two or more aggregates of the mature adipocytes, each of which forms lipid droplets with a diameter of 20 μm or greater.