Artificial fat tissue and method for producing the same, method for producing artificial skin and agent for adipocyte culture

Abstract

To provide an artificial adipose tissue capable of being maintained for a long period of time and to provide a production method capable of producing the artificial adipose tissue in a short period; to provide an artificial skin including an artificial adipose tissue capable of being maintained for a long period of time and to provide a production method capable of producing the artificial skin in a short period; and to provide an agent for adipocyte culture and to provide a method for evaluating skin permeability of a compound by using the artificial skin.SOLUTION: The present invention provides an artificial adipose tissue comprising a three-dimensional tissue comprising: cells comprising adipocytes; and a fragmented extracellular matrix.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to artificial adipose tissue and a method for producing the same, a method for producing artificial skin, and a culture medium for adipocytes. [Background technology]

[0002] Long-term in vitro culture maintenance of mature adipocytes is a critical challenge, particularly in the fields of cosmetic / pharmaceutical assays or cosmetic surgery. Conventional two-dimensional culture on flat dishes has a short culture time, and adipocytes mainly dedifferentiate after one week. Existing reports indicate that monocular mature adipocytes cannot be maintained for more than one week (Non-Patent Literature 1). Furthermore, adipocytes that have matured to a certain extent tend to float away from the culture dish due to the influence of lipid droplets within the cells, making it difficult to change the culture medium.

[0003] To address these challenges, three-dimensional cell culture technology is attracting attention. WAQAR HASSAN et al. have encapsulated cells in a hydrogel composed of a PEG-based copolymer and hyaluronic acid and are culturing them (Non-Patent Literature 2). However, this method isolates and cultures the cells, making physical contact between cells difficult and resulting in poor differentiation efficiency.

[0004] DAQUINAG et al. have demonstrated the simultaneous simulation of angiogenesis and lipidogenesis using a three-dimensional structured culture system based on magnetic nanoparticles, employing mouse 3T3-L1 preadipocytes and GFP-expressing mouse endothelial cells. While co-culture of adipocytes and endothelial cells appears well-preserved by immunostaining, the adipocytes after 14 days are very small and show low maturity (Non-Patent Literature 3).

[0005] On the other hand, regarding skin models containing adipocytes, there are reports of a two-layer skin model with fibroblasts and adipocytes using bovine type I collagen hydrogel (3 mg / mL), but the culture required to construct the skin model is extremely long, ranging from 56 to 63 days. Furthermore, its thickness is thinner than that of human skin (Non-Patent Literature 4).

[0006] Furthermore, there are reports of skin models using fibroblasts resuspended in human frozen plasma hydrogel (1 mg / mL fibrinogen), or a three-layer structure of mesenchymal stem cells and adipocytes. However, these models require a long culture process of 35 days to construct, and they do not morphologically mimic the structure of human skin, being very thin (Non-Patent Literature 5). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Toda S, Uchihashi K, Aoki S, et al., Organogenesis. 2009 5(2):50-56 [Non-Patent Document 2] WAQAR HASSAN ET AL., STEM CELL RESEARCH & THERAPY 2013,21;4(2):32 [Non-Patent Document 3] DAQUINAG, ET AL., TISSUE ENGINEERING. PART C, METHODS, 2013May;19(5):336-44. [Non-Patent Document 4] TROTTIER ET AL., STEM CELLS, 2008 Oct;26 (10): 2713-23 [Non-Patent Document 5] MONFORT ET AL., J Tissue Eng Regen Med., 2013 Jun;7(6):479-90 [Overview of the project] [Problems that the invention aims to solve]

[0008] One embodiment of the present invention aims to provide artificial adipose tissue that can be maintained for a long period of time and a method for producing said artificial adipose tissue in a short time. Another embodiment of the present invention aims to provide artificial skin containing artificial adipose tissue that can be maintained for a long period of time and a method for producing said artificial skin in a short time. Another embodiment of the present invention aims to provide a culture medium for adipose cells and a method for evaluating the skin permeability of compounds using the above-mentioned artificial skin. [Means for solving the problem]

[0009] The inventors have discovered that by culturing adipocytes, which are suspended cells, together with fragmented extracellular matrix to form a three-dimensional tissue, adipocytes can be differentiated in a short time, and artificial adipose tissue composed of the formed three-dimensional tissue can be maintained for a long period of time. Furthermore, they have found that by using this artificial adipose tissue, it is possible to manufacture artificial skin with a thickness close to that of human skin.

[0010] In other words, the present invention provides, for example, the following [1] to

[29] . [1] Artificial adipose tissue consisting of a three-dimensional tissue body containing cells including adipocytes and fragmented extracellular matrix. [2] The artificial adipose tissue described in [1], wherein the extracellular matrix described above contains collagen. [3] The artificial adipose tissue according to [1] or [2], wherein the content of the extracellular matrix is ​​10% to 30% by weight, based on the artificial adipose tissue. [4] Artificial adipose tissue as described in any of [1] to [3], with a thickness of 1 mm to 10 mm. [5] Artificial adipose tissue as described in any of [1] to [4], wherein the shrinkage rate during culture is 20% or less. [6] The artificial adipose tissue according to any one of [1] to [5], wherein the average length of the fragmented extracellular matrix is 100 nm to 200 μm. [7] The artificial adipose tissue according to any one of [1] to [6], wherein the average value of the size of the lipid droplets of the adipocytes is 20 μm to 180 μm. [8] An artificial skin comprising a first layer and a second layer, wherein the first layer is made of the artificial adipose tissue according to any one of [1] to [7]. [9] The artificial skin according to [8], wherein the second layer contains fibroblasts.

[10] The artificial skin according to [8] or [9], further comprising a third layer.

[11] The artificial skin according to

[10] , wherein the third layer contains keratinocytes.

[12] The artificial skin according to any one of [8] to

[11] , having a thickness of 0.2 mm to 10 mm.

[13] (1) A step of contacting cells containing adipocytes with a fragmented extracellular matrix in an aqueous medium, and (2) A step of culturing the cells contacted with the fragmented extracellular matrix, a method for producing an artificial adipose tissue.

[14] The production method according to

[13] , wherein the culturing time in step (2) is 10 days to 30 days.

[15] The production method according to

[13] or

[14] , further comprising a step of co-precipitating the fragmented extracellular matrix in the aqueous medium and the cells between step (1) and step (2).

[16] The production method according to any one of

[13] to

[15] , wherein the extracellular matrix contains collagen.

[17] The production method according to any one of

[13] to

[16] , wherein the average length of the fragmented extracellular matrix is 100 nm to 200 μm.

[18] (1) In an aqueous medium, bring into contact a first cell containing adipocytes with a first fragmented extracellular matrix, and (2) A step of forming a first layer, which includes culturing the first cells in contact with the first fragmented extracellular matrix, (3) A step of forming a second layer, which includes further contacting the first layer with second cells and culturing the second cells. A method for manufacturing artificial skin, including

[19] A method for producing artificial skin according to

[18] , wherein the average length of the first fragmented extracellular matrix is ​​100 nm to 200 μm.

[20] The manufacturing method according to

[18] or

[19] , wherein the incubation time in step (2) is 10 to 60 days. [twenty one] A method for producing artificial skin according to any one of

[18] to

[20] , wherein step (3) includes bringing the second cells into contact with the fragmented second extracellular matrix on the first layer in an aqueous medium and culturing the second cells. [twenty two] The method for producing artificial skin according to

[21] , wherein the second extracellular matrix described above contains collagen. [twenty three] A method for producing artificial skin according to any one of

[18] to

[22] , wherein the second cell described above includes fibroblasts. [twenty four] A step to form a third layer, which includes further contacting the second layer with third cells and culturing the third cells. A method for producing artificial skin according to any one of

[18] to

[23] , further comprising: [twenty five] The method for producing artificial skin according to

[24] , wherein the third cell described above includes keratinocytes.

[26] A culture medium for adipocytes containing fragmented extracellular matrix.

[27] The culture medium described in

[26] , wherein the fragmented extracellular matrix is ​​collagen with an average length of 100 nm to 200 μm.

[28] A culture medium according to

[26] or

[27] , which is a differentiation promoter.

[29] A method for evaluating the skin permeability of a compound, The process of preparing artificial skin as described in any of [8] to

[12] , The process involves bringing the test compound into contact with artificial skin, A step of measuring the skin permeability of the test compound using artificial skin that has been contacted with the test compound, A step of comparing the skin permeability of the above test compound with a reference value. Methods that include... [Effects of the Invention]

[0011] The present invention makes it possible to provide artificial adipose tissue that can be maintained for a long period of time and a method for producing said artificial adipose tissue in a short time. Furthermore, the present invention makes it possible to provide artificial skin containing artificial adipose tissue that can be maintained for a long period of time and a method for producing said artificial skin in a short time. Furthermore, the present invention makes it possible to provide a culture medium for adipose cells and a method for evaluating the skin permeability of compounds using the above-mentioned artificial skin.

[0012] Furthermore, according to the present invention, it becomes possible to provide artificial skin with a thickness close to that of human skin, and a method for manufacturing the same. [Brief explanation of the drawing]

[0013] [Figure 1] This is a schematic diagram illustrating the manufacturing process of artificial adipose tissue, which consists of a three-dimensional tissue composed of fragmented collagen and mature adipocytes or adipose stem cells. [Figure 2]This photograph shows the results of evaluating the artificial adipose tissue from Example 1 on day 14 of culture. A shows the results of hematoxylin-eosin (HE) staining. The arrows indicate an example of the presence of fragmented collagen. The dotted lines indicate an example of mature adipocytes. B shows the results of Nile Red lipid staining. The white areas indicate nuclei. C shows the results using a live / dead kit. Gray indicates viable cells, and white indicates dead cells. [Figure 3] These are photographs showing the results of immunostaining with anti-perilipin antibodies for artificial adipose tissue from Example 1 and artificial adipose tissue produced by conventional two-dimensional culture. The white areas indicate nuclei. In the photographs, 3D shows the results for artificial adipose tissue from Example 1, and 2D shows the results for artificial adipose tissue produced by conventional two-dimensional culture. [Figure 4] This graph shows a comparison of the size of adipose tissue vesicles in the artificial adipose tissue of Example 1 and artificial adipose tissue produced by conventional two-dimensional culture on days 7 and 14 of culture (*p<0.001). In the graph, 3D represents the results for the artificial adipose tissue of Example 1, and 2D represents the results for artificial adipose tissue produced by conventional two-dimensional culture. [Figure 5] This photograph shows the results of HE staining of artificial adipose tissue from Example 2 on day 17 of differentiation. The upper photograph is a magnified portion of the lower photograph, with the dotted line indicating an example of mature adipocytes and the arrow indicating an example of a location where fragmented collagen is present. [Figure 6] These are electron microscope images of artificial adipose tissue produced by conventional two-dimensional culture at days 37 and 72 of differentiation. The top image shows artificial adipose tissue at day 37 of differentiation, and the bottom image shows artificial adipose tissue at day 72 of differentiation. The arrows indicate an example of mature adipocytes. [Figure 7] This photograph shows the results of immunostaining with anti-perilipin antibodies for artificial adipose tissue from Example 2 and artificial adipose tissue produced by conventional two-dimensional culture. The white areas represent nuclei. In the photograph, 3D shows the results for artificial adipose tissue from Example 2, and 2D shows the results for artificial adipose tissue produced by conventional two-dimensional culture. [Figure 8]This graph shows a comparison of the size of adipose tissue vesicles in the artificial adipose tissue of Example 2 and the artificial adipose tissue produced by conventional two-dimensional culture on days 7 and 14 of culture (*p<0.001). In the graph, 3D represents the results for the artificial adipose tissue of Example 2, and 2D represents the results for the artificial adipose tissue produced by conventional two-dimensional culture. [Figure 9] This is a schematic diagram illustrating the manufacturing process of artificial skin, which includes a first layer consisting of artificial adipose tissue containing fragmented collagen and mature adipocytes, a second layer containing fibroblasts, and a third layer containing keratinocytes. [Figure 10] This photograph shows the results of HE staining of artificial skin (left) prepared using 1 × 10⁶ adipocytes and 10 mg of fragmented collagen in Example 3, and subcutaneous tissue skin of a mouse (right), on day 9 of culture (day 7 of keratinocyte differentiation). [Figure 11] These are photographs showing the results of HE staining of artificial skin from Example 3, which was prepared using 1 × 10⁶ adipocytes and 10 mg of fragmented collagen, on day 9 of culture (day 7 of keratinocyte differentiation). A is a magnified view of the layer containing keratinocytes, B is a magnified view of the layer containing fibroblasts, and C is a magnified view of the layer containing adipocytes. [Figure 12] These are photographs showing the results of HE staining of artificial skin from Example 3, which was prepared using 5 × 10⁵ adipocytes and 10 mg of fragmented collagen, on day 9 of culture (day 7 of keratinocyte differentiation). A is a magnified view of the layer containing keratinocytes, B is a magnified view of the layer containing fibroblasts, and C is a magnified view of the layer containing adipocytes. [Figure 13] These are photographs showing the results of HE staining of artificial skin from Example 3, which was prepared using 1 × 10⁶ adipocytes and 15 mg of fragmented collagen, on day 9 of culture (day 7 of keratinocyte differentiation). A is a magnified view of the layer containing keratinocytes, B is a magnified view of the layer containing fibroblasts, and C is a magnified view of the layer containing adipocytes. [Figure 14]This graph shows the relationship between keratinocyte differentiation time and the thickness of the artificial skin in the three-layered artificial skin manufactured in Example 3. The black squares represent the results for the artificial skin of Example 3 manufactured using 5 × 10⁵ adipocytes and 10 mg of fragmented collagen, the black circles represent the results for the artificial skin of Example 3 manufactured using 1 × 10⁶ adipocytes and 10 mg of fragmented collagen, and the white circles represent the results for the artificial skin of Example 3 manufactured using 1 × 10⁶ adipocytes and 15 mg of fragmented collagen. [Figure 15] The expression of adipogenic genes PPARγ2, FABP4, and GLUT4 in three-dimensional human adipose stem cell tissue (3D) and artificial adipose tissue (2D) produced by conventional two-dimensional culture up to 21 days of differentiation was evaluated using RT-qPCR, normalized by the expression level of the housekeeping gene RPII. The results are shown below. Asterisks indicate statistical significance compared to two-dimensional tissue (*p<0.05, **p<0.01, and ***p<0.001). [Modes for carrying out the invention]

[0014] (artificial adipose tissue) The artificial adipose tissue according to this embodiment consists of a three-dimensional tissue body comprising cells including adipocytes and fragmented extracellular matrix. At least a portion of the cells are adhered to the fragmented extracellular matrix. Compared to artificial adipose tissue produced by conventional two-dimensional culture on a flat dish, the artificial adipose tissue according to this embodiment exhibits enhanced differentiation and can be maintained for a long period of time.

[0015] The artificial adipose tissue according to this embodiment is artificially created adipose tissue and does not include adipose tissue itself that has been simply isolated from living tissue.

[0016] A "three-dimensional tissue" refers to an aggregate of cells in which cells are arranged three-dimensionally via an extracellular matrix such as collagen, and is an aggregate artificially created by cell culture. There are no particular restrictions on the shape of the three-dimensional tissue; for example, it can be sheet-shaped, spherical, ellipsoidal, or rectangular. In this embodiment, the artificial adipose tissue consists of a three-dimensional tissue containing fragmented extracellular matrix, and can therefore be distinguished from biological tissue and three-dimensional tissues produced by other methods that do not use fragmented extracellular matrix, based on the presence or absence of fragmented extracellular matrix.

[0017] In this embodiment, "adipocytes" include not only differentiated cells such as mature adipocytes, but also undifferentiated cells such as adipose stem cells. Adipocytes may be cells collected from, for example, subcutaneous adipose tissue, epicardial adipose tissue, etc., or collected cells may be differentiated and used. Adipocytes are not particularly limited, but when adipose tissue constructed from adipocytes is ultimately used to represent tissue in a specific part of the living body, it is preferable to use adipocytes derived from tissue corresponding to that part. Examples of animal species from which adipocytes can be derived include humans, mice, rats, and pigs. Preferred adipocytes include mature adipocytes or adipose stem cells.

[0018] "Extracellular matrix" refers to substances that exist outside of cells in living organisms, and specifically includes collagen, elastin, proteoglycans, fibronectin, hyaluronic acid, etc. In this embodiment, the extracellular matrix is ​​preferably a substance that exists outside of animal cells, i.e., the extracellular matrix of an animal, more preferably contains collagen or elastin, even more preferably collagen or elastin, and particularly preferably collagen.

[0019] Examples of collagen include fibrous collagen and non-fibrous collagen. Fibrous collagen refers to collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. An example of non-fibrous collagen is type IV collagen.

[0020] Conventional three-dimensional tissues have low concentrations of extracellular matrix such as collagen and high cell density. As a result, problems such as the three-dimensional tissue shrinking due to the traction force of cells during or after culture, or the three-dimensional tissue being easily degraded by enzymes produced by cells during or after culture, have occurred. In addition, although porous high-density collagen is commercially available, it is not possible to uniformly adhere collagen to cells with these materials, and it is difficult to recover cells for subsequent evaluation of cell characteristics. The adipose tissue composed of the three-dimensional tissue according to this embodiment has a higher extracellular matrix content than conventional three-dimensional tissues, making it less prone to shrinkage and more stable.

[0021] The extracellular matrix content in the three-dimensional tissue may be 0.01 to 90% by weight, preferably 10 to 90% by weight, more preferably 1 to 50% by weight, more preferably 10 to 30% by weight, and particularly preferably 20 to 30% by weight, based on the three-dimensional tissue. Here, "extracellular matrix in the three-dimensional tissue" refers to the extracellular matrix constituting the three-dimensional tissue, and may be endogenous or exogenous extracellular matrix. Furthermore, "extracellular matrix in the three-dimensional tissue" also includes fragmented extracellular matrix, which will be described later. That is, if the three-dimensional tissue contains endogenous extracellular matrix, the concentration of the extracellular matrix constituting the three-dimensional tissue refers to the combined concentration of the endogenous extracellular matrix and the fragmented extracellular matrix. The concentration of the extracellular matrix can be calculated from the volume of the obtained three-dimensional tissue and the mass of the decellularized three-dimensional tissue.

[0022] "Endogenous extracellular matrix" refers to the extracellular matrix produced by extracellular matrix-producing cells, while "endogenous collagen" refers to the collagen produced by collagen-producing cells that constitute three-dimensional tissues. Endogenous collagen may be fibrous collagen or non-fibrous collagen.

[0023] "Exogenous extracellular matrix" refers to extracellular matrix supplied from an external source. The artificial adipose tissue according to this embodiment consists of a three-dimensional tissue body, which includes fragmented extracellular matrix. The exogenous extracellular matrix may be derived from the same or different animal species as the endogenous extracellular matrix. Examples of the animal species include humans, pigs, and cattle. The exogenous extracellular matrix may also be an artificial extracellular matrix. Fragmented extracellular matrix is ​​exogenous extracellular matrix. When the extracellular matrix is ​​collagen, it is also called "exogenous collagen," meaning collagen supplied from an external source, and specifically includes fibrous collagen, non-fibrous collagen, etc. The exogenous collagen is preferably fibrous collagen. Examples of the above-mentioned fibrous collagen include type I collagen, type II collagen, and type III collagen, with type I collagen being preferred. The above-mentioned fibrous collagen may be commercially available collagen, and a specific example is freeze-dried type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. An example of exogenous non-fibrous collagen is type IV collagen.

[0024] In exogenous extracellular matrix, the animal species from which it originates may differ from that of the cells. Furthermore, if the cells include extracellular matrix-producing cells, the animal species from which the exogenous extracellular matrix originates may differ from that of the extracellular matrix-producing cells. In other words, the exogenous extracellular matrix may be a heterogeneous extracellular matrix.

[0025] The above three-dimensional tissue contains fragmented extracellular matrix. "Fragmented extracellular matrix" means extracellular matrix such as collagen that has been fragmented. The extracellular matrix from which the fragmented extracellular matrix originates may be one type or multiple types of extracellular matrix may be used in combination. Conventionally, extracellular matrix such as collagen was dissolved in an acidic aqueous solution, but the concentration was only about 0.1 to 0.3% by weight, so it was not possible to dissolve a large amount. Therefore, it was difficult to increase the amount of extracellular matrix such as collagen in the three-dimensional tissue using conventional methods. The fragmented extracellular matrix according to this embodiment is almost insoluble in water, but it is presumed that by dispersing it in an aqueous medium described later, it will come into contact with cells including adipocytes in the aqueous medium, thereby promoting the formation of the three-dimensional tissue. The average length of the fragmented extracellular matrix 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 extracellular matrix is ​​preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0026] When the extracellular matrix is ​​collagen, the fragmented extracellular matrix is ​​also called "fragmented collagen." "Fragmented collagen" refers to collagen, such as fibrous collagen, that has been fragmented while maintaining a triple helix structure. The average length of the fragmented collagen 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 is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0027] There are no particular limitations on the method for fragmenting extracellular matrix such as collagen. For example, the extracellular matrix may be fragmented using a homogenizer such as an ultrasonic homogenizer, agitator homogenizer, or high-pressure homogenizer. When using an agitator homogenizer, the extracellular matrix may be homogenized directly or in an aqueous medium such as physiological saline. Furthermore, by adjusting the homogenization time, number of repetitions, etc., it is possible to obtain fragmented extracellular matrix of millimeter or nanometer size.

[0028] The diameter and length of fragmented extracellular matrix can be determined by analyzing individual fragments of extracellular matrix using an electron microscope.

[0029] According to the manufacturing method of this embodiment, artificial adipose tissue consisting of a large three-dimensional tissue body with a thickness of 1 mm or more can be produced with a relatively small number of cells.

[0030] The artificial adipose tissue composed of the above three-dimensional tissue preferably has a thickness of 10 μm to 20 mm, more preferably 100 μm to 15 mm, and particularly preferably 1 mm to 10 mm. The lower limit of the thickness is not particularly limited, but may be, for example, 10 μm, 50 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1 mm, 3 mm, or 5 mm. The upper limit of the thickness is not particularly limited, but may be, for example, 20 mm, 15 mm, 10 mm, 5 mm, 3 mm, 2 mm, 1.5 mm, or 1 mm. Such a three-dimensional tissue has a structure closer to biological tissue and is suitable as a substitute for experimental animals and as a transplant material.

[0031] Here, "thickness of the three-dimensional structure" means the distance between the two ends in a direction perpendicular to the main surface when the three-dimensional structure is in the form of a sheet or a rectangular parallelepiped. If the main surface has irregularities, the thickness means the distance at the thinnest part of the main surface. If the three-dimensional structure is spherical, it means its diameter. Furthermore, if the three-dimensional structure is ellipsoidal, it means its minor axis. If the three-dimensional structure is roughly spherical or ellipsoidal and has irregularities on its surface, the thickness means the shortest distance between two points where a line passing through the centroid of the three-dimensional structure intersects the surface.

[0032] The artificial adipose tissue composed of the three-dimensional tissue described above preferably has a shrinkage rate of 20% or less during culture, more preferably 15% or less, and even more preferably 10% or less. The shrinkage rate can be calculated, for example, by the following formula. In the formula, L1 represents the length of the longest part of the artificial adipose tissue composed of the three-dimensional tissue on day 1 after culture, and L3 represents the length of the corresponding part of the artificial adipose tissue composed of the three-dimensional tissue on day 3 after culture. Contraction rate (%) = {(L1 - L3) / L1} × 100 In the example above, the contraction rate is calculated from artificial adipose tissue on day 1 and day 3 after culturing. However, it may also be calculated from artificial adipose tissue on day 1 and day 2 after culturing, from artificial adipose tissue on day 1 and day 5 after culturing, or from artificial adipose tissue on day 1 and day 8 after culturing.

[0033] The size of lipid droplets can be used as an indicator of the maturity of cultured adipocytes. Lipid droplets are intracellular organelles that store lipids such as triglycerides and cholesterol, and have a droplet-like shape because these lipids are covered with a single membrane of phospholipids. Furthermore, the surface of these phospholipids shows expression of proteins specific to adipose tissue (such as perilipin). Although there is variability in the size of lipid droplets in mature adipocytes, for example, if the average size of lipid droplets is 20 μm or more, the adipocytes can be considered to be somewhat mature, i.e., mature adipocytes.

[0034] In the artificial adipose tissue of this embodiment, the average size of the lipid droplets of adipocytes is preferably 20 μm to 180 μm, and more preferably 100 μm to 180 μm. The size of the lipid droplets may be 250 μm or less, 200 μm or less, 180 μm or less, or 150 μm or less. The size of the lipid droplets may also be 15 μm or more, 30 μm or more, 50 μm or more, or 80 μm or more. The size of the lipid droplets may also be the value obtained on day 7 of culture, day 10 of culture, day 14 of culture, or day 21 of culture.

[0035] Furthermore, expression profiles of gene markers such as adipocyte formation markers and lipolysis markers can be used as indicators of the maturity of cultured adipocytes. Examples of adipocyte formation markers include Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2), Fatty Acid Binding Protein 4 (FABP4), and Glucose transporter type 4 (GLUT-4). PPARγ2 and FABP4 can be used as early markers of adipocyte formation, while GLUT-4 can be used as a late marker. Examples of lipolysis markers include hormone-sensitive lipase (HSL) and Adipocyte Triglyceride Lipase (ATGL).

[0036] PPARγ2 is one of the most important transcription factors in adipocyte differentiation. During adipogenesis, PPARγ2 expression has been reported to increase, then remain constant or decrease (B. Galateanu et al., Int. J. Mol. Sci. 2012 (13) 15881-15900 and A. Soukas et al., J. Biol. Chem. 2001 (276) 34167-34174). Therefore, for example, tissues with constant PPARγ2 expression are thought to be in a more advanced stage of adipogenesis compared to tissues with increasing PPARγ2. FABP4 is another early marker required for the transport of fatty acids to the membrane for efflux. Therefore, for example, tissues showing a more typical linear increase in FABP4 expression profile are thought to be in an earlier stage of adipogenesis. For a typical linear increase in FABP4 expression profile, see, for example, ECM Mariman et al., Cell. Mol. Life Sci. 2010 (67) 1277-1292.

[0037] Insulin-stimulated GLUT-4 is the major glucose transporter in adipocytes. GLUT-4 expression is weak in tissues where adipogenesis is in its early stages (S.-W. Qian et al., BMC Dev. Biol. 2010 (10) 47). Therefore, for example, tissues with earlier increases in GLUT-4 expression are thought to have more advanced adipogenesis.

[0038] The artificial adipose tissue of this embodiment may contain cells other than adipocytes, and may also contain extracellular matrix-producing cells. "Extracellular matrix-producing cells" means cells that secrete extracellular matrix such as collagen. In other words, the artificial adipose tissue may contain endogenous extracellular matrix. Examples of extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, and fibroblasts are preferred. Examples of preferred fibroblasts include human skin-derived fibroblasts (NHDF), human cardiac fibroblasts (NHCF), and human gingival fibroblasts (HGF). Other types of cells besides adipocytes include, for example, vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVEC)), colorectal cancer cells (e.g., human colorectal cancer cells (HT29)), cancer cells such as liver cancer cells, cardiomyocytes (e.g., human iPS cell-derived cardiomyocytes (iPS-CM)), epithelial cells (e.g., human gingival epithelial cells), lymphatic endothelial cells, nerve cells, hepatocytes, tissue stem cells, embryonic stem cells, induced pluripotent stem cells, adherent cells (e.g., immune cells), smooth muscle cells (e.g., aortic smooth muscle cells (Arota-SMC)), and keratinocytes (e.g., human epidermal keratinocytes).

[0039] The adipose tissue composed of the above three-dimensional tissue can be used as a substitute for experimental animals, a transplant material, etc. Specifically, it can be used, for example, in cosmetic assay screening for cellulite and obesity, screening of pharmaceuticals for diabetes and obesity, in vitro models of other pathological conditions such as inflammatory diseases related to adipose tissue, and tissue reconstruction after soft tissue defects caused by trauma or tumor removal, or after mastectomy.

[0040] (artificial skin) The artificial skin according to this embodiment includes a plurality of layers, one of which is a layer made of the artificial adipose tissue described above. That is, the artificial skin according to this embodiment includes a first layer and a second layer, and includes the artificial adipose tissue described above as the first layer. The artificial skin only needs to include at least the first layer and the second layer, and may further include a third layer, a fourth layer or a fifth layer, or even more layers. The order of the layers does not matter, but it is preferable that they be stacked in the order of the first layer, the second layer, and the third layer. However, even in this case, it is sufficient that the first layer, the second layer, and the third layer are in this order, and it is not limited to including another layer below the first layer, between the first and second layers, between the second and third layers, or above the third layer.

[0041] Each layer of the artificial skin according to this embodiment can be distinguished based on differences in the types of cells it contains and their distribution. For example, by staining the artificial skin with different colors for each type of cell, each layer will be shown in a different color, allowing it to be distinguished by cross-sectional photographs, etc. Alternatively, for example, all the cells contained in the artificial skin can be stained, and each layer can be distinguished by differences in the density of stained cells in cross-sectional photographs, etc.

[0042] The layers other than the first layer (the second, third, fourth, and fifth layers, etc.) contain cells. These cells may include the adipocytes mentioned above, or they may include cells other than adipocytes. The second layer preferably contains fibroblasts or keratinocytes, more preferably fibroblasts, and even more preferably human skin-derived fibroblasts or human cardiac fibroblasts. The third layer preferably contains fibroblasts or keratinocytes, more preferably keratinocytes, and even more preferably human epidermal keratinocytes. When the first layer is the bottom layer, it is preferable that the second layer contains fibroblasts and the third layer contains keratinocytes. This layering allows for the creation of artificial skin that more closely resembles the skin structure in the human body.

[0043] Furthermore, layers other than the first layer (the second, third, fourth, and fifth layers, etc.) may contain extracellular matrix or fragmented extracellular matrix. It is preferable that the second layer contains fragmented extracellular matrix. The same extracellular matrix and fragmented extracellular matrix as described above can be used. By including fragmented extracellular matrix, it is possible to form layers with appropriate spacing between cells.

[0044] The artificial skin according to this embodiment preferably has a thickness of 10 μm to 20 mm, more preferably 100 μm to 15 mm, even more preferably 200 μm to 10 mm, and particularly preferably 1 mm to 10 mm. The lower limit of the thickness is not particularly limited, but may be, for example, 10 μm, 50 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1 mm, 3 mm, or 5 mm. The upper limit of the thickness is not particularly limited, but may be, for example, 20 mm, 15 mm, 10 mm, 5 mm, 3 mm, 2 mm, 1.5 mm, or 1 mm. Such artificial skin has a structure closer to biological tissue and is suitable as a substitute for experimental animals and as a transplant material.

[0045] "Artificial skin thickness" refers to the distance from the surface of the uppermost tissue layer to the bottom of the lowermost tissue layer. For example, in the case of a three-layer tissue including epithelial, dermal, and subcutaneous tissue, it refers to the distance from the surface of the epithelial tissue to the bottom of the subcutaneous tissue (the total thickness of the three layers).

[0046] The number of cells constituting the artificial skin according to this embodiment is 1 × 10 6 There may be more than one.

[0047] The first layer of the artificial skin in this embodiment consists of the artificial adipose tissue described above. In the artificial adipose tissue, the average size of the lipid droplets of the adipocytes is preferably 20 μm to 180 μm, and more preferably 100 μm to 180 μm. The size of the lipid droplets may be 250 μm or less, 200 μm or less, 180 μm or less, or 150 μm or less. The size of the lipid droplets may also be 15 μm or more, 30 μm or more, 50 μm or more, or 80 μm or more. The size of the lipid droplets may also be the value on day 7 of culture, day 10 of culture, day 14 of culture, or day 21 of culture.

[0048] The above-mentioned artificial skin can be used as a substitute for living skin, as a substitute for experimental animals, as a transplant material, etc. Specifically, it can be used, for example, in tests to evaluate the skin permeability, skin safety, skin corrosion, and skin irritation of compounds, in cosmetic assay screening, in pharmaceutical screening, in vitro pathological models, and as a transplant material for orthopedic surgery and burn surgery.

[0049] (Method of manufacturing artificial adipose tissue) The method for producing artificial adipose tissue according to this embodiment is: (1) A step of bringing cells containing adipocytes into contact with fragmented extracellular matrix in an aqueous medium (hereinafter also referred to as step (1)), and (2) The process includes the step of culturing the cells that have come into contact with the fragmented extracellular matrix (hereinafter also referred to as step (2)).

[0050] The terms "adipocytes," "fragmented extracellular matrix," etc., are as described above.

[0051] In this embodiment, the "cells including adipocytes" in step (1) may include cells other than adipocytes, and may also include extracellular matrix-producing cells. "Extracellular matrix-producing cells" means cells that secrete extracellular matrix such as collagen. Examples of extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, and fibroblasts are preferred. Examples of preferred fibroblasts include human skin-derived fibroblasts (NHDF), human cardiac fibroblasts (NHCF), and human gingival fibroblasts (HGF). Other types of cells besides adipocytes include, for example, vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVEC)), colorectal cancer cells (e.g., human colorectal cancer cells (HT29)), cancer cells such as liver cancer cells, cardiomyocytes (e.g., human iPS cell-derived cardiomyocytes (iPS-CM)), epithelial cells (e.g., human gingival epithelial cells), lymphatic endothelial cells, nerve cells, hepatocytes, tissue stem cells, embryonic stem cells, induced pluripotent stem cells, adherent cells (e.g., immune cells), smooth muscle cells (e.g., aortic smooth muscle cells (Arota-SMC)), and keratinocytes (e.g., human epidermal keratinocytes).

[0052] An "aqueous medium" refers to a liquid in which water is an essential component. There are no particular restrictions on the aqueous medium, as long as it allows fragmented collagen and cells to exist stably. Examples include salines such as phosphate-buffered saline (PBS), Dulbecco's Modified Eagle medium (DMEM), and vascular endothelial cell medium (EGM2). The liquid medium may also be a mixed medium, a mixture of two different media. From the viewpoint of reducing the burden on cells, the aqueous medium is preferably a liquid medium.

[0053] There are no particular limitations on the method for bringing fragmented extracellular matrix into contact with cells, including adipocytes, in an aqueous medium. For example, this could involve adding a dispersion of fragmented extracellular matrix to a culture medium containing cells, adding cells to a culture medium dispersion of fragmented extracellular matrix, or adding fragmented extracellular matrix and cells separately to a pre-prepared aqueous medium.

[0054] The concentration of fragmented extracellular matrix in the aqueous medium in step (1) can be appropriately determined according to the shape, thickness, and size of the culture vessel of the artificial adipose tissue consisting of the target three-dimensional tissue. For example, the concentration of fragmented extracellular matrix in the aqueous medium in step (1) may be 0.1 to 90% by weight, or 1 to 30% by weight.

[0055] The amount of fragmented extracellular matrix in step (1) is 1 × 10⁻⁶ 5 The amount may be 0.1 to 100 mg or 1 to 50 mg relative to the cells.

[0056] In step (1), the mass ratio of the fragmented extracellular matrix to the cells is preferably 1000:1 to 1:1, more preferably 900:1 to 9:1, and even more preferably 500:1 to 10:1.

[0057] When using both adipocytes and other cells, the ratio (number of cells) of adipocytes to other cells in step (1) may be 99:1 to 9:1 or 80:20 to 50:50.

[0058] The process may further include a step between steps (1) and (2) in which cells, including adipocytes, and fragmented extracellular matrix in an aqueous medium are allowed to settle together. Performing such a step results in a more uniform distribution of fragmented extracellular matrix and cells in the artificial adipose tissue composed of a three-dimensional tissue. There are no particular limitations on the specific method, but one example is a method of centrifuging a culture medium containing fragmented extracellular matrix and cells.

[0059] Step (1) may be performed by forming a layer of cells in an aqueous medium and then bringing it into contact with the fragmented extracellular matrix. By forming the layer of cells before bringing it into contact with the fragmented extracellular matrix, it is possible to create artificial adipose tissue consisting of a three-dimensional tissue with a high cell density in the lower layer. For example, by forming a layer of cells containing extracellular matrix-producing cells before bringing it into contact with the fragmented extracellular matrix, it is possible to create artificial adipose tissue consisting of a three-dimensional tissue with a high cell density in the lower layer of cells containing extracellular matrix-producing cells. Depending on the type of cells used, this method can be used to create artificial adipose tissue that is closer to that of living organisms.

[0060] In this embodiment, the method for producing a three-dimensional tissue may include, after step (2), a step (3) in which cells are further brought into contact and cultured. The cells may be the same as or different from the cells used in step (1). For example, if the cells used in step (1) include cells other than extracellular matrix-producing cells, the cells used in step (3) may include extracellular matrix-producing cells. Also, for example, if the cells used in step (1) include extracellular matrix-producing cells, the cells used in step (3) may include cells other than extracellular matrix-producing cells. Both the cells used in step (1) and the cells used in step (3) may include extracellular matrix-producing cells, or both the cells used in step (1) and the cells used in step (3) may include cells other than extracellular matrix-producing cells. By step (3), an artificial adipose tissue consisting of a two-layered three-dimensional tissue can be produced. Artificial skin containing multiple layers can be produced by a similar method. The method for producing said artificial skin will be described later.

[0061] In step (2), the method for culturing cells that have come into contact with the fragmented extracellular matrix is ​​not particularly limited and can be performed using a suitable culture method depending on the type of cells to be cultured. For example, the culture temperature may be 20°C to 40°C or 30°C to 37°C. The pH of the culture medium may be 6 to 8 or 7.2 to 7.4. The manufacturing method according to this embodiment does not require the complex composition of culture media used in conventional two-dimensional culture and three-dimensional tissue production, and can use easily prepared media such as DMEM. The culture medium is not particularly limited, and a suitable medium can be selected depending on the type of cells to be cultured. Examples of media include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The culture medium may be a serum-added medium or a serum-free medium. The culture medium may be a mixed medium obtained by mixing two types of media. Furthermore, according to the manufacturing method according to this embodiment, well-differentiated artificial adipose tissue can be obtained in a much shorter culture time compared to conventional two-dimensional culture. Therefore, the culture time in step (2) may be, for example, 10 to 60 days, 10 to 30 days, 13 to 25 days, or 14 to 17 days. However, the above culture times only indicate the time required to obtain sufficiently differentiated adipose tissue and do not prevent further culture.

[0062] The cell density in the culture medium in step (2) can be appropriately determined according to the shape, thickness, and size of the culture vessel of the target three-dimensional tissue. For example, the cell density in the culture medium in step (2) can be 1 to 10 8 It may be cells / mL, 10 3 ~10 7 The cell density may be cells / mL. Also, the cell density in the culture medium in step (2) may be the same as the cell density in the aqueous medium in step (1).

[0063] The artificial adipose tissue, consisting of a three-dimensional tissue body produced by the manufacturing method according to this embodiment, preferably has a shrinkage rate of 20% or less during culture, more preferably 15% or less, and even more preferably 10% or less. The above shrinkage rate can be calculated, for example, by the following formula. In the formula, L1 represents the length of the longest part of the three-dimensional tissue body on day 1 after culture, and L3 represents the length of the corresponding part of the three-dimensional tissue body on day 3 after culture. Contraction rate (%) = {(L1 - L3) / L1} × 100 In the example above, the contraction rate is calculated from the artificial adipose tissue on day 1 and day 3 after culturing. However, it may also be calculated from the artificial adipose tissue at any point during the culturing period, including the end of culturing. For example, it may be calculated from the artificial adipose tissue on day 1 and day 2 after culturing, from the artificial adipose tissue on day 1 and day 5 after culturing, or from the artificial adipose tissue on day 1 and day 8 after culturing.

[0064] The artificial adipose tissue consisting of a three-dimensional tissue body produced by the manufacturing method according to this embodiment preferably has a retention rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more after trypsin treatment at a trypsin concentration of 0.25%, a temperature of 37°C, a pH of 7.4, and a reaction time of 15 minutes. Such a three-dimensional tissue body is stable and less susceptible to enzymatic degradation during or after culture. The above retention rate can be calculated, for example, from the mass of the three-dimensional tissue body before and after trypsin treatment.

[0065] The artificial adipose tissue consisting of a three-dimensional tissue body produced by the manufacturing method according to this embodiment preferably has a residual rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more after collagenase treatment at a collagenase concentration of 0.25%, a temperature of 37°C, a pH of 7.4, and a reaction time of 15 minutes. Such a three-dimensional tissue body is stable and less susceptible to enzymatic degradation during or after culture.

[0066] Furthermore, the manufacturing method according to this embodiment makes it possible to produce artificial adipose tissue consisting of a stable three-dimensional tissue body in which cells are uniformly distributed. Moreover, compared to conventional two-dimensional culture, the artificial adipose tissue produced by the manufacturing method according to this embodiment maintains mature adipocytes for a longer period of time. In addition, the manufacturing method according to this embodiment makes it possible to produce artificial adipose tissue consisting of a large three-dimensional tissue body with a thickness of 1 mm or more using a relatively small number of cells.

[0067] (Method of manufacturing artificial skin) The method for manufacturing artificial skin according to this embodiment is: (1) In an aqueous medium, bring into contact a first cell containing adipocytes with a first fragmented extracellular matrix (hereinafter also referred to as step (1)'), and (2) A step of forming a first layer (hereinafter also referred to as step (2)') which includes culturing the first cells that have come into contact with the first fragmented extracellular matrix, (3) A step of forming a second layer (hereinafter also referred to as step (3)') which includes further contacting the first layer with second cells and culturing the second cells. Includes.

[0068] For steps (1)' and (2)', the same methods as those described above (method for producing artificial adipose tissue) can be used. The aqueous medium, the first cells containing adipocytes, the second cells, and the first fragmented extracellular matrix can also be the same as those described above.

[0069] The method for forming the first layer in step (2)' is not particularly limited, but the layer can be considered formed when the thickness of the cultured cells reaches the desired thickness. It is not necessary to start step (3)' after the formation of the first layer in step (2)' is complete; step (3)' can be started during the culture in step (2)'.

[0070] The average length of the first fragmented extracellular matrix 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 extracellular matrix is ​​preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0071] Furthermore, according to the manufacturing method of this embodiment, well-differentiated adipose tissue (first layer) can be obtained in a much shorter culture time compared to conventional two-dimensional culture. Therefore, the culture time in step (2)' may be, for example, 10 to 60 days, 10 to 30 days, 13 to 25 days, or 14 to 17 days. However, the above culture time merely indicates the time required to obtain well-differentiated adipose tissue and does not prevent further culture.

[0072] The second cells may be the same as or different from the first cells. The methods for bringing the cells into contact and culturing them are also the same as those described above (method for producing artificial adipose tissue).

[0073] The cells used as the second layer are the same as those described above (artificial adipose tissue), but it is preferable that the second layer contains fibroblasts. By making the second layer a layer differentiated from fibroblasts, a structure closer to that of living skin can be achieved.

[0074] Furthermore, it is preferable that step (3)' includes bringing the second cells into contact with the fragmented second extracellular matrix in an aqueous medium on the first layer and culturing the second cells. The second extracellular matrix can be the same as that described above in (artificial adipose tissue). By including the fragmented extracellular matrix, a layer with appropriate spacing between cells can be formed. It is preferable that the second extracellular matrix contains collagen, and more preferably that the second extracellular matrix is ​​collagen.

[0075] The method for producing artificial skin according to this embodiment may further include a step of forming a third layer, which involves bringing a third cell into contact with the second layer and culturing the third cell.

[0076] The cells used as the third layer are the same as those described above (artificial adipose tissue), but it is preferable that the third layer contains keratinocytes. By making the third layer a layer of differentiated keratinocytes, a structure closer to that of living skin can be achieved.

[0077] According to the method for producing artificial skin of this embodiment, artificial skin with a thickness close to that of living skin can be obtained in a much shorter culture time compared to conventional two-dimensional culture, similar to the method described above (artificial skin).

[0078] Furthermore, according to the manufacturing method of artificial skin according to this embodiment, it is possible to manufacture artificial skin that includes, as a first layer, artificial adipose tissue consisting of a stable, three-dimensional tissue body in which cells are uniformly distributed. Moreover, the artificial adipose tissue in the artificial skin manufactured by the manufacturing method according to this embodiment maintains mature adipocytes for a longer period of time compared to conventional two-dimensional culture. In addition, according to the manufacturing method according to this embodiment, it is possible to manufacture artificial skin with a thickness of 1 mm or more, which is close to the thickness of living skin, with a relatively small number of cells.

[0079] (Adipose-derived cell culture medium) The adipocyte culture medium according to this embodiment includes fragmented extracellular matrix. The adipocyte culture medium according to this embodiment may have an average length of 100 nm to 200 μm of the fragmented extracellular matrix, and an average diameter of 50 nm to 30 μm of the fragmented extracellular matrix. Furthermore, regarding the length of the fragmented extracellular matrix, 95% of the entire fragmented extracellular matrix may be in the range of 100 nm to 200 μm. In addition, regarding the diameter of the fragmented extracellular matrix, 95% of the entire fragmented extracellular matrix may be in the range of 50 nm to 30 μm.

[0080] "Adipose cell culture medium" refers to a reagent for culturing adipocytes. The adipose cell culture medium may be in powder form or in the form of a dispersion in which fragmented extracellular matrix is ​​dispersed in an aqueous medium. The type, size, manufacturing method, and method of using the culture medium are the same as those shown in (Artificial Adipose Tissue) and (Method for Manufacturing Artificial Adipose Tissue) above. The inclusion of fragmented extracellular matrix promotes the differentiation of adipocytes. Therefore, the adipose cell culture medium can also be considered a differentiation promoter. Furthermore, the inclusion of fragmented extracellular matrix makes dedifferentiation of mature adipocytes less likely. Therefore, the adipose cell culture medium can also be considered a dedifferentiation inhibitor of mature adipocytes (a maintenance agent for mature adipocytes). Moreover, according to the culture medium of this embodiment, even when forming tissue with a thickness of 1 mm or more, for example, it is possible to form tissue with little tissue shrinkage due to long-term culture, thus enabling the formation of tissue with stable morphology for a long period of time while exhibiting differentiation-promoting and dedifferentiation-inhibiting effects.

[0081] (Method for evaluating the skin permeability of compounds) A method for evaluating the skin permeability of the compound according to this embodiment is: The process of preparing artificial skin, The process involves bringing the test compound into contact with artificial skin, A step of measuring the skin permeability of the test compound using artificial skin that has been contacted with the test compound, A step of comparing the skin permeability of the above test compound with a reference value. This includes the artificial skin described above (Artificial Skin) or the artificial skin manufactured by the manufacturing method described above (Method for Manufacturing Artificial Skin). As described above, since the artificial skin has a similar structure and thickness to living skin, it can be used as a substitute for living skin in skin permeability tests of compounds.

[0082] There are no particular restrictions on the test compound; any compound commonly used in skin permeability tests can be used.

[0083] There are no particular limitations on the method of contacting the test compound with the artificial skin. For example, if the test compound is a liquid, it can be directly applied or sprayed onto the surface of the artificial skin. If the test compound is a solid, a solution obtained by dissolving it in a solvent can be used to similarly apply or spray it onto the surface of the artificial skin.

[0084] The skin permeability of a test compound can be measured based on skin permeability testing methods well known to those skilled in the art. For example, safety evaluations for cosmetics and quasi-drugs can be conducted in accordance with the guidance in Pharmaceutical and Medical Devices Agency Notification No. 1115-1 (November 15, 2016).

[0085] The reference value can be set appropriately based on the type and concentration of the test compound, etc. For example, if the reference value is one point, a value higher than the reference value can be evaluated as high skin permeability, and a value lower than the reference value can be evaluated as low skin permeability. Alternatively, the reference value can be set as a range for evaluating skin permeability in stages.

[0086] The present invention will be described in more detail and specifically below with reference to examples, but these examples are not intended to limit the scope of the present invention. [Examples]

[0087] (Example 1: Production of artificial adipose tissue using mature mouse adipocytes) The inventors developed a method for producing three-dimensional tissue using collagen microfibers to increase collagen density in the tissue (Figure 1). Artificial adipose tissue using mature adipocytes was manufactured as follows, as shown in the schematic diagram in Figure 1.

[0088] Freeze-dried type I collagen derived from porcine skin, manufactured by Nippon Ham Co., Ltd., was dispersed in 10x concentrated phosphate-buffered saline (×10 PBS) and homogenized for 5 minutes using a homogenizer to obtain fragmented collagen with a diameter of approximately 4.4 μm and a length of approximately 22.5 μm. The obtained fragmented collagen was washed with serum-free medium (DMEM) to obtain a culture medium dispersion of fragmented collagen.

[0089] Fragmented collagen was dispersed in a serum-containing culture medium (DMEM) to a concentration of 10 mg / mL. 1 mL of the resulting dispersion (equivalent to approximately 10 mg of fragmented collagen) was mixed with 1 × 10⁶ of the DMEM. 6Primary mature mouse adipocytes were mixed with cells in a 24-well Transwell plate (IWAKI). 24 hours after seeding, the Transwell mixture was placed in a 6-well plate (IWAKI) containing 6 mL of culture medium (DMEM medium, Nacalai Tesque). The mature adipocytes were cultured for 14 days, with the culture medium changed every 4 days. At the start of culture, only a mixture containing cells and fragmented collagen accumulated; no centrifugation was performed. As the culture progressed, the thickness in the direction of self-weight decreased and stabilized, and after 14 days of culture, a three-dimensional tissue body approximately 2 mm thick was obtained. The artificial adipose tissue composed of the obtained three-dimensional tissue body was stained with hematoxylin-eosin (HE) staining and / or immunostaining with anti-perilipin antibody for histological evaluation. Lipids were also stained using Nile Red. Survival rates were evaluated using a live / dead kit (Thermo Fisher Scientific), and the survival / death and analysis of adipocyte vesicles were performed using ImageJ software (National Institutes of Health). Furthermore, the diameters of 100 lipid vesicles were measured using an electron microscope on days 7 and 14 of culture, and compared with the adipocytes of artificial adipose tissue produced by conventional two-dimensional culture.

[0090] The conventional method for producing artificial adipose tissue using two-dimensional culture was performed as follows: 3 x 10⁶ cells were placed in a 6-well plate (manufactured by IWAKI Corporation). 5 Primary mature mouse adipocytes were seeded (cells / well) and cultured. Culture was continued in 2 mL of culture medium (DMEM medium, Nacalai Tesque). The medium was changed every 3 days. Since mature adipocytes do not adhere to the bottom of the container due to the buoyancy of lipid droplets, a commercially available coverslip was floated in the medium, and the cells were cultured by adhering them to the bottom of the coverslip.

[0091] HE staining revealed the presence of densely packed, fragmented collagen within the three-dimensional tissue surrounding mature adipocytes (Figure 2A, arrow). The mature adipocytes exhibited a typical round, simple morphology with small cytoplasm and nuclei (Figure 2A).

[0092] Lipid staining with Nile Red revealed lipid droplets within the cytoplasm. Monocular lipid droplets, indicating mature adipocytes, were observed, demonstrating homogeneous and highly refractionated mature adipocytes within the three-dimensional tissue (Figure 2B). Even after long-term culture of 14 days or more, the artificial adipose tissue maintained mature adipocytes well with a high survival rate of 98.5% on day 2 to 94.6% on day 14 (Figure 2C; gray indicates viable cells, white indicates dead cells). Immunostaining with anti-perilipin antibody confirmed that mature adipocytes in the three-dimensional tissue at day 14 maintained a larger monocular shape (54±8 μm lipid vesicles on day 0) compared to adipocytes in artificial adipose tissue produced by conventional two-dimensional culture (Figure 3). On the other hand, in artificial adipose tissue produced by conventional two-dimensional culture, mature adipocytes dedifferentiated into fibroblast-like cell morphology and showed vesicles that were significantly 3.7 times smaller than those of mature adipocytes in three-dimensional tissue on day 14 of culture (Figure 4, counting n>100 adipocyte vesicles, t-test). Furthermore, in artificial adipose tissue composed of three-dimensional tissue containing fragmented collagen, the size of adipocyte vesicles was maintained from day 7 to day 14, while in artificial adipose tissue produced by conventional two-dimensional culture, the size of adipocyte vesicles decreased from day 7 to day 14. This indicates that three-dimensional tissue containing fragmented collagen can be maintained for a longer period because it takes longer to dedifferentiate compared to artificial adipose tissue produced by conventional two-dimensional culture, and that the culture time of mature adipocytes could be extended by at least one week.

[0093] (Example 2: Production of artificial adipose tissue using human adipose stem cells) Artificial adipose tissue using adipose stem cells was manufactured as follows, as shown in the schematic diagram in Figure 1.

[0094] Freeze-dried type I collagen derived from porcine skin, manufactured by Nippon Ham Co., Ltd., was dispersed in 10x concentrated phosphate-buffered saline (×10 PBS) and homogenized for 5 minutes using a homogenizer to obtain fragmented collagen with a diameter of approximately 4.4 μm and a length of approximately 22.5 μm. The obtained fragmented collagen was washed with serum-free medium (DMEM) to obtain a culture medium dispersion of fragmented collagen.

[0095] Fragmented collagen was dispersed in a medium containing serum (DMEM) at a concentration of 10 mg / mL. 300 μL of the resulting dispersion (equivalent to about 3 mg of fragmented collagen) and 5×10 5 human adipose-derived stem cells in cells were mixed in a 96-well insert (manufactured by ACEA Bioscience). After 24 hours of seeding, 200 μL of a medium (DMEM medium, manufactured by Nacalai Tesque) was added. The medium was changed every two days, and the cells were cultured for 19 days. Of the 19 days, 2 days corresponded to the growth period, and the subsequent 17 days corresponded to the differentiation period. Since only the mixture containing cells and fragmented collagen had accumulated at the start of the culture and no centrifugation was performed, the thickness in the direction of its own weight decreased over the course of the culture and became stable, and a three-dimensional tissue mass with a thickness of about 2 mm was obtained after 14 days of culture. The artificial adipose tissue composed of the three-dimensional tissue mass on the 17th day of differentiation (19th day of culture) was stained with hematoxylin and eosin (HE) and / or immunostained with an anti-perilipin antibody for histological evaluation.

[0096] HE staining showed that there was high-density fragmented collagen in the three-dimensional tissue mass (arrow in Fig. 5). Also, large round monocular-shaped lipid droplets indicating mature adipocytes were confirmed (enlarged view in Fig. 5). Among them, mature adipocytes with a size exceeding 100 μm were also confirmed.

[0097] The method for producing artificial adipose tissue by conventional two-dimensional culture was performed as follows. 1×10 4 (cells / well) human adipose-derived stem cells were seeded and cultured in a 24-well plate (manufactured by IWAKI). For the first two days after seeding, the cells were cultured using 500 μL of D-MEM to promote growth, and then the medium was changed to a medium for promoting differentiation. The medium for promoting differentiation was D-MEM supplemented with PGM-2 Singlequot Kit PT-9502 (manufactured by LONZA) and contained growth supplements (Indomethacin, 3-isobutyl-1-methylxanthine, dexamethasone, insulin). The cells were cultured with 500 μL, and the medium was changed once a week.

[0098] Artificial adipose tissue produced by conventional two-dimensional culture still showed scattered lipid vesicles in droplet form even at 37 days of differentiation (Figure 6). Furthermore, lipid staining with Nile Red revealed only small lipid droplets, and no mature adipocytes larger than 100 μm were observed, unlike in three-dimensional tissues containing fragmented collagen.

[0099] Furthermore, in artificial adipose tissue produced by conventional two-dimensional culture, droplet-like fat vesicles were still observed even at 72 days of differentiation, indicating that not all adipocytes had differentiated (Figure 6). In addition, as the amount of fat increased, the adipocytes separated and floated in the culture medium, making it difficult to change the culture medium.

[0100] Immunostaining with anti-perilipin antibodies revealed that adipose stem cells from three-dimensional tissue at day 7 of culture formed larger monocular shapes (4 μm ± 2 μm adipose vesicles on day 7) compared to adipose stem cells from artificial adipose tissue produced by conventional two-dimensional culture (Figure 7). On the other hand, artificial adipose tissue produced by conventional two-dimensional culture showed vesicles that were significantly twice as small as those in adipose cells within three-dimensional tissue at day 7 of culture (Figure 8, counting n>100 adipose vesicles, t-test). Furthermore, the size of adipose vesicles in artificial adipose tissue consisting of three-dimensional tissue containing fragmented collagen more than doubled (10 μm ± 4 μm) from day 7 to day 14, while the size of adipose vesicles in artificial adipose tissue produced by conventional two-dimensional culture hardly changed from day 7 to day 14.

[0101] The above results demonstrate that artificial adipose tissue composed of a three-dimensional tissue structure containing fragmented collagen differentiates more rapidly than artificial adipose tissue produced by conventional two-dimensional culture.

[0102] (Example 3: Production of artificial skin using mature adipocytes) Artificial skin using mature adipocytes was manufactured as follows, as shown in the schematic diagram in Figure 9.

[0103] Freeze-dried type I collagen derived from porcine skin, manufactured by Nippon Ham Co., Ltd., was dispersed in 10x concentrated phosphate-buffered saline (×10 PBS) and homogenized for 5 minutes using a homogenizer to obtain fragmented collagen with a diameter of approximately 4.4 μm and a length of approximately 22.5 μm. The obtained fragmented collagen was washed with serum-free medium (DMEM) to obtain a culture medium dispersion of fragmented collagen.

[0104] Fragmented collagen was dispersed in a serum-containing culture medium (DMEM) to a concentration of 10 mg / mL. 1 mL of the resulting dispersion (equivalent to approximately 10 mg of fragmented collagen) was mixed with 1 × 10¹⁶ fragments of collagen collected from rat subcutaneous tissue in DMEM. 6 Mature rat adipocytes from cells were mixed in a 24-well Transwell plate (IWAKI Corporation). The plate was placed in an incubator and cultured for 24 hours.

[0105] Next, the 24-well inserts were coated with 0.04 mg / mL fibronectin solution (#F2006-5G, Sigma) in PBS (0.04 μL / insert) and incubated at 37°C for 20 minutes. Subsequently, 1 mL of the above fragmented collagen medium dispersion (equivalent to approximately 10 mg of fragmented collagen) and 5 × 10⁻¹⁴ 5 Human skin-derived fibroblasts (NHDF) from cells were mixed, and 100 μL was distributed into a 24-well insert. The plate was placed in an incubator and cultured for 24 hours.

[0106] Next, the culture medium in the insert was aspirated onto the NHDF collagen gel, coated with 0.04 mg / mL collagen IV solution in PBS (0.04 μL / insert), and incubated at 37°C for at least 20 minutes. The collagen IV solution added to the NHDF collagen gel was aspirated, and 2 × 10⁻⁶ samples were placed on the NHDF collagen gel. 6Normal human epidermal keratinocytes (#KK-4009, 1 vial = 500,000 cells, KURABO) were added. 1 mL of DMEM 5% FBS:EpiLife (#C-2517A, Invitrogen) (1:1) medium was added to the outside of the insert, and another 1 mL was added to the outside of the insert after 1 hour.

[0107] The inner and outer culture media were gently aspirated, and 500 μL of differentiation medium, diluted 100-fold with ascorbic acid in DMEM 5% FBS:EpiLife (1:1) medium, was added to the outside of the insert. No medium was added to the inside of the insert. The medium outside the insert was replaced daily until day 7 of differentiation.

[0108] According to the above method, artificial skin with a three-layer structure—a layer of adipose tissue at the bottom, a layer of fibroblasts in the middle, and a layer of keratinocytes at the top—can be produced in just 9 days from the start of adipose tissue culture (24 hours of adipose tissue culture + 24 hours of fibroblast culture + 7 days of keratinocyte differentiation) (Figure 9).

[0109] Artificial skin cells produced as described above, at day 7 of differentiation, were stained with hematoxylin and eosin (HE) and histologically evaluated. The artificial skin cells showed appropriate intercellular spacing in all layers, including the lower layer containing adipocytes (subcutaneous tissue), the middle layer containing fibroblasts (dermal tissue), and the upper layer containing keratinocytes (epidermal tissue), demonstrating a structure similar to in vivo skin tissue (Figures 10 and 11). The keratinocytes formed an epithelial-like layer, and enucleation, one of the morphological indicators of keratinocyte differentiation, was also observed (Figure 11). Furthermore, the artificial skin cells at day 7 of differentiation had a thickness (distance from the surface of the epithelial tissue to the bottom surface of the lowest layer of subcutaneous tissue) of approximately 4.3 mm, demonstrating that the thickness closely resembled that of in vivo skin tissue.

[0110] The adipocytes used to produce adipose tissue are 1 × 10⁶ 6 From cells 5 × 10 5When the cells were changed, and when the amount of fragmented collagen used was changed from 10 mg to 15 mg, the same results as above were observed (Figures 12 and 13). Figure 14 shows the relationship between the differentiation time of keratinocytes and the thickness of the three-layered artificial skin. It was shown that the decrease in skin thickness subsided and stagnation occurred between days 3 and 7 of keratinocyte differentiation.

[0111] (Example 4: Promotion of adipogenesis by adipose stem cells in three-dimensional tissue) Similar to Example 2, the expression of adipogenesis genes in three-dimensional tissues containing fragmented collagen produced using human adipose stem cells and artificial adipose tissues produced by conventional two-dimensional culture was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) (Figure 15).

[0112] Total RNA was extracted from the three-dimensional tissues and artificial adipose tissues produced by conventional two-dimensional culture using the PureLink RNA Microkit (Invitrogen) according to the kit protocol. The extracted RNA was quantified using Nanodrop® N1000 (ThermoFisher Scientific). 1 μg of RNA was converted to cDNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems) according to the kit protocol. PCR was performed by amplifying 70 ng of cDNA with 0.3 μM forward and reverse primers using iTaq® Universal SYBR Green Supermix (BioRAD). The sequences of each primer used, the optimal number of amplification cycles for RT-PCR, and the temperature conditions are shown in Table 1. cDNA synthesis and RT-qPCR reactions were performed using the StepOnePlus® Real-Time PCR System (ThermoFisher Scientific).

[0113] [Table 1]

[0114] Adipogenesis involves a transition from the proliferation stage to the differentiation stage, starting with the expression of initial adipogenesis genes (e.g., FABP4, PPARγ2) when preadipocytes become immature adipocytes, followed by the expression of late-stage genes (e.g., GLUT4) as lipids accumulate in lipid vesicles. While the expression of both initial and late-stage markers was found to gradually increase throughout the culture period, differences in expression were observed between three-dimensional tissue and artificial adipose tissue produced by two-dimensional culture. The amount of mRNA in the three-dimensional tissue containing the aforementioned fragmented collagen tended to be higher than in artificial adipose tissue produced by two-dimensional culture, with the expression of the initial gene FABP4 being up to 5.5 times higher on day 7 and the expression of the late-stage gene GLUT4 being 8.3 times higher on day 21 (Figure 15).

[0115] PPARγ2 gene expression continued to increase in artificial adipose tissue produced by two-dimensional culture, but in the three-dimensional tissue, although it showed a slightly enhanced expression profile from day 7 to day 21, it remained almost constant. In the three-dimensional tissue, PPARγ2 expression was already at a constant stage, suggesting that adipogenesis was more advanced. Another early marker, FABP4, remained generally constant in the three-dimensional tissue from day 7 to day 21. On the other hand, an increase in expression was observed in the artificial adipose tissue produced by two-dimensional culture from day 7 to day 21. Expression of insulin-stimulated GLUT-4, a late marker, increased sharply from day 14 in the three-dimensional tissue, while GLUT-4 expression was observed very weakly in the artificial adipose tissue produced by two-dimensional culture. These results indicate that the three-dimensional tissue exhibits gene expression that is more in line with the differentiation state compared to the artificial adipose tissue produced by two-dimensional culture.

Claims

1. An artificial adipose tissue comprising a three-dimensional tissue comprising cells including adipocytes and fragmented extracellular matrix, the average length of the fragmented extracellular matrix being 22 μm to 200 μm.

2. The artificial adipose tissue described in claim 1, wherein the extracellular matrix contains collagen.

3. An artificial adipose tissue as described in claim 1 or 2, having a thickness of 200 μm to less than 10 mm.

4. An artificial adipose tissue described in any one of claims 1 to 3, wherein the fragmented extracellular matrix is homogenized fragmented collagen.

5. An artificial adipose tissue described in any one of claims 1 to 4, wherein the fragmented extracellular matrix is dispersible in an aqueous medium.

6. Artificial skin comprising a first layer and a second layer, the first layer consisting of the artificial adipose tissue described in any one of claims 1 to 5.

7. The artificial skin described in claim 6, wherein the second layer contains fibroblasts.

8. Artificial skin as described in claim 6 or 7, further comprising a third layer, said third layer comprising keratinocytes.

9. Artificial skin described in any one of claims 6 to 8, having a thickness of more than 200 μm to 10 mm.

10. (1) A method for producing a cell-derived cell matrix comprising contacting cells, including adipocytes, with fragmented extracellular matrix in an aqueous medium; and (2) culturing the cells in contact with the fragmented extracellular matrix; A method for producing artificial adipose tissue, wherein the average length of the fragmented extracellular matrix is 22 μm to 200 μm.

11. The manufacturing method described in claim 10, wherein the extracellular matrix comprises collagen.

12. A manufacturing method described in claim 10 or 11, wherein the fragmented extracellular matrix is homogenized fragmented collagen.

13. A manufacturing method described in any one of claims 10 to 12, wherein the fragmented extracellular matrix is dispersible in an aqueous medium. (1) contacting first cells, including adipocytes, with a first fragmented extracellular matrix in an aqueous medium; and (2) forming a first layer, the step including culturing the first cells in contact with the first fragmented extracellular matrix; (3) forming a second layer, which further includes contacting second cells with the first layer and culturing the second cells; A method for producing artificial skin, wherein the average length of the fragmented extracellular matrix is 22 μm to 200 μm.

15. A method for producing artificial skin as described in claim 14, wherein the first fragmented extracellular matrix is homogenized fragmented collagen.

16. A method for producing artificial skin as described in claim 14 or 15, wherein step (3) comprises contacting the second cells with a fragmented second extracellular matrix in an aqueous medium on the first layer and culturing the second cells, and the second extracellular matrix comprises collagen.

17. A method for producing artificial skin as described in claim 14 or 15, wherein the second cells include fibroblasts.

18. A method for producing artificial skin described in any one of claims 14 to 17, further comprising a step of forming a third layer, which includes contacting third cells with the second layer and culturing the third cells, wherein the third cells include keratinocytes.

19. A manufacturing method described in any one of claims 14 to 18, wherein the fragmented extracellular matrix is dispersible in an aqueous medium.

20. An agent for promoting differentiation of adipocytes, comprising fragmented extracellular matrix, the average length of the fragmented extracellular matrix being 22 μm to 200 μm.

21. The differentiation promoter described in claim 20, wherein the fragmented extracellular matrix is homogenized fragmented collagen.

22. A method for assessing the skin permeability of a compound, comprising: A step of preparing the artificial skin according to any one of claims 6 to 9, and a step of contacting a test compound with the artificial skin; measuring the skin permeability of the test compound using the artificial skin contacted with the test compound; and comparing the skin permeability of the test compound with a reference value.

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