Artificial adipose tissue and its manufacturing method, artificial skin manufacturing method, and culture agent for adipocytes

Abstract

Disclosed is an artificial adipose tissue comprising a three-dimensional tissue structure containing cells, including adipocytes, and fragmented extracellular matrix.

Description

[Technical field]

[0001] The present invention relates to an artificial adipose tissue and a method for producing the same, a method for producing artificial skin, and a culture agent for adipocytes. [Background technology]

[0002] Long-term in vitro culture and maintenance of mature adipocytes is one of the important issues, especially in the field of cosmetic / pharmaceutical assays or plastic surgery. Conventional two-dimensional culture on a flat dish has a short culture time, and adipocytes mainly dedifferentiate after one week. According to existing reports, monocular mature adipocytes cannot be maintained for more than one week (Non-Patent Document 1). In addition, adipocytes that have matured to a certain degree tend to float off the culture dish due to the influence of lipid droplets within the cells, making it difficult to replace the culture medium.

[0003] To solve these problems, three-dimensional cell culture technology has attracted attention. Waqar Hassan et al. have encapsulated cells in a hydrogel consisting of a PEG-based copolymer and hyaluronic acid and are culturing them (Non-Patent Document 2). However, this method cultures cells in isolation, making it physically difficult for cells to come into contact with each other, and differentiation efficiency is poor.

[0004] have demonstrated that a magnetic nanoparticle-based three-dimensional structure culture system can simultaneously simulate angiogenesis and adipogenesis using mouse 3T3-L1 preadipocytes and GFP-expressing mouse endothelial cells. Although the co-culture of adipocytes and endothelial cells appears beautiful by immunostaining, the fat vesicles after 14 days are very small and low in maturity (Non-Patent Document 3).

[0005] On the other hand, regarding a skin model containing fat cells, a two-layered skin model of fibroblasts and fat stem cells using bovine type I collagen hydrogel (3 mg / mL) has been reported, but the culture for constructing the skin model requires a very long period of time, 56 to 63 days, and the thickness is also thinner than that of human skin (Non-Patent Document 4).

[0006] There have also been reports of skin models with a three-layer structure consisting of fibroblasts resuspended in human frozen plasma hydrogel (1 mg / mL fibrinogen) or mesenchymal stem cells and adipocytes; however, the model requires a long culture process of 35 days to construct, and is very thin and does not morphologically mimic the human skin structure (Non-Patent Document 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, 2013 May;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 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of one embodiment of the present invention is to provide an artificial adipose tissue that can be maintained for a long period of time, and to provide a method for producing the artificial adipose tissue in a short period of time.An object of one embodiment of the present invention is further to provide an artificial skin containing an artificial adipose tissue that can be maintained for a long period of time, and to provide a method for producing the artificial skin in a short period of time.An object of one embodiment of the present invention is further to provide a culture agent for adipocytes, and to provide a method for evaluating the skin permeability of a compound using the artificial skin. [Means for solving the problem]

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

[0010] That is, the present invention provides, for example, the following [1] to

[29] . [1] An artificial adipose tissue consisting of a three-dimensional tissue structure containing cells including adipocytes and fragmented extracellular matrix. [2] The artificial adipose tissue described in [1], wherein the extracellular matrix contains collagen. [3] The artificial adipose tissue according to [1] or [2], wherein the content of the extracellular matrix is ​​10% by weight to 30% by weight based on the artificial adipose tissue. [4] The artificial adipose tissue described in any one of [1] to [3], having a thickness of 1 mm to 10 mm. [5] The artificial adipose tissue according to any one of [1] to [4], which has a contraction rate during culture of 20% or less. [6] The artificial adipose tissue described in any 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 size of the lipid droplets in the fat cells is 20 μm to 180 μm. [8] An artificial skin comprising a first layer and a second layer, the first layer being made of the artificial adipose tissue according to any one of [1] to [7]. [9] The artificial skin described in [8], wherein the second layer contains fibroblasts.

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

[11] The artificial skin described in

[10] , wherein the third layer contains keratinocytes.

[12] The artificial skin described in any one of [8] to

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

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

[14] The method according to

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

[15] The manufacturing method according to

[13] or

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

[16] The method according to any one of

[13] to

[15] , wherein the extracellular matrix contains collagen.

[17] The 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) contacting a first cell, including an adipocyte, with a first fragmented extracellular matrix in an aqueous medium; and (2) forming a first layer comprising culturing the first cells in contact with the first fragmented extracellular matrix; (3) forming a second layer, the step including contacting second cells with the first layer and culturing the second cells; A method for producing artificial skin comprising:

[19] The 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 method according to

[18] or

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

[18] to

[20] , wherein step (3) comprises contacting the second cells with fragmented second extracellular matrix in an aqueous medium on the first layer and culturing the second cells. [twenty two] The method for producing artificial skin described in

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

[18] to

[22] , wherein the second cells include fibroblasts. [twenty four] forming a third layer, the step further comprising contacting third cells with the second layer and culturing the third cells; The method for producing artificial skin according to any one of

[18] to

[23] , further comprising: [twenty five] The method for producing artificial skin described in

[24] , wherein the third cells include keratinocytes.

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

[27] The culture agent according to

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

[28] The culture agent according to

[26] or

[27] , which is a differentiation promoter.

[29] A method for evaluating the skin permeability of a compound, comprising: A step of preparing an artificial skin according to any one of [8] to

[12] ; contacting a test compound with an artificial skin; measuring the skin permeability of the test compound using an artificial skin contacted with the test compound; comparing the skin permeability of the test compound with a reference value; A method comprising: Effect of the Invention

[0011] According to the present invention, it is possible to provide artificial adipose tissue that can be maintained for a long period of time, and to provide a manufacturing method that can produce the artificial adipose tissue in a short period of time.Furthermore, according to the present invention, it is possible to provide artificial skin containing artificial adipose tissue that can be maintained for a long period of time, and to provide a manufacturing method that can produce the artificial skin in a short period of time.Furthermore, according to the present invention, it is possible to provide a culture agent for adipocytes, and to provide a method for evaluating the skin permeability of a compound using the artificial skin.

[0012] Furthermore, according to the present invention, it is possible to provide artificial skin having a thickness close to that of human skin and a method for producing the same. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing the manufacturing process of an artificial adipose tissue consisting of a three-dimensional tissue structure containing fragmented collagen and mature adipocytes or adipose stem cells. [Diagram 2]Photographs showing the results of evaluating the artificial adipose tissue of Example 1 on the 14th day of culture. A shows the results of hematoxylin-eosin (HE) staining. The arrow indicates an example of a location where fragmented collagen is present. The dotted line indicates an example of a mature adipocyte. B shows the results of Nile red lipid staining. The white areas indicate nuclei. C shows the results using the Live / Dead kit. Grey indicates live cells, and white indicates dead cells. [Diagram 3] Photographs showing the results of immunostaining with an anti-perilipin antibody for the artificial adipose tissue of Example 1 and the artificial adipose tissue produced by conventional two-dimensional culture. The white areas indicate nuclei. In the photographs, 3D shows the results for the artificial adipose tissue of Example 1, and 2D shows the results for the artificial adipose tissue produced by conventional two-dimensional culture. [Figure 4] 1 is a graph showing the results of comparing the size of fat 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 shows the results for the artificial adipose tissue of Example 1, and 2D shows the results for the artificial adipose tissue produced by conventional two-dimensional culture. In the graph, 3D shows the results for the artificial adipose tissue of Example 1, and 2D shows the results for the artificial adipose tissue produced by conventional two-dimensional culture. [Diagram 5] Photographs showing the results of HE staining of the artificial adipose tissue of Example 2 on day 17 of differentiation. The upper photograph is an enlarged portion of the lower photograph, with the dotted line indicating an example of a mature adipocyte and the arrow indicating an example of a location where fragmented collagen is present. [Figure 6] These are electron microscope photographs of artificial adipose tissue produced by conventional two-dimensional culture on days 37 and 72 of differentiation. The upper photograph shows the artificial adipose tissue on day 37 of differentiation, and the lower photograph shows the artificial adipose tissue on day 72 of differentiation. The arrows indicate an example of a mature adipocyte. [Figure 7] Photographs showing the results of immunostaining with an anti-perilipin antibody for the artificial adipose tissue of Example 2 and the artificial adipose tissue produced by conventional two-dimensional culture. The white areas indicate nuclei. In the photographs, 3D shows the results for the artificial adipose tissue of Example 2, and 2D shows the results for the artificial adipose tissue produced by conventional two-dimensional culture. [Figure 8]1 is a graph showing the results of comparing the size of fat vesicles in the artificial adipose tissue of Example 2 and artificial adipose tissue produced by conventional two-dimensional culture on days 7 and 14 of culture (*p<0.001). In the graph, 3D shows the results for the artificial adipose tissue of Example 2, and 2D shows the results for the artificial adipose tissue produced by conventional two-dimensional culture. [Figure 9] This is a schematic diagram showing the manufacturing process of artificial skin comprising 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] These photographs show the results of HE staining of the artificial skin of Example 3 (left) produced using 1 x 106 fat cells and 10 mg of fragmented collagen, and mouse subcutaneous tissue skin (right) on the 9th day of culture (7th day of differentiation of keratinocytes). [Figure 11] Photographs showing the results of HE staining of the artificial skin of Example 3 produced using 1×106 fat cells and 10 mg of fragmented collagen on the 9th day of culture (7th day of differentiation of keratinocytes). A is a magnified photograph of the layer containing keratinocytes, B is a magnified photograph of the layer containing fibroblasts, and C is a magnified photograph of the layer containing fat cells. [Figure 12] Photographs showing the results of HE staining of the artificial skin of Example 3 produced using 5×105 fat cells and 10 mg of fragmented collagen on the 9th day of culture (7th day of differentiation of keratinocytes). A is a magnified photograph of the layer containing keratinocytes, B is a magnified photograph of the layer containing fibroblasts, and C is a magnified photograph of the layer containing fat cells. [Figure 13] Photographs showing the results of HE staining of the artificial skin of Example 3 produced using 1×106 fat cells and 15 mg of fragmented collagen on the 9th day of culture (7th day of differentiation of keratinocytes). A is a magnified photograph of the layer containing keratinocytes, B is a magnified photograph of the layer containing fibroblasts, and C is a magnified photograph of the layer containing fat cells. [Figure 14]1 is a graph showing the relationship between the differentiation time of keratinocytes and the thickness of the artificial skin with a three-layer structure produced in Example 3. The black squares show the results of the artificial skin of Example 3 produced using 5×105 cells of fat cells and 10 mg of fragmented collagen, the black circles show the results of the artificial skin of Example 3 produced using 1×106 cells of fat cells and 10 mg of fragmented collagen, and the white circles show the results of the artificial skin of Example 3 produced using 1×106 cells of fat cells and 15 mg of fragmented collagen. [Figure 15] The expression of adipogenic genes PPARγ2, FABP4, and GLUT4 was evaluated by RT-qPCR up to 21 days of differentiation in three-dimensional human adipose stem cell tissue (3D) and artificial adipose tissue (2D) produced by conventional two-dimensional culture, normalized by the expression level of the housekeeping gene RPII. Asterisks indicate significant differences compared to 2D tissue (*p<0.05, **p<0.01, and ***p<0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] (artificial adipose tissue) The artificial adipose tissue according to the present embodiment is a three-dimensional tissue comprising cells, including adipocytes, and a fragmented extracellular matrix. At least a portion of the cells are attached to the fragmented extracellular matrix. The artificial adipose tissue according to the present embodiment has promoted differentiation and can be maintained for a long period of time, compared to artificial adipose tissue produced by conventional two-dimensional culture on a flat dish.

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

[0016] The term "three-dimensional tissue" refers to an assembly of cells in which cells are arranged three-dimensionally via an extracellular matrix such as collagen, and is artificially produced by cell culture. The shape of the three-dimensional tissue is not particularly limited, and examples of the shape include a sheet, a sphere, an ellipsoid, and a rectangular prism. Here, the artificial adipose tissue of the present embodiment is made of a three-dimensional tissue containing a fragmented extracellular matrix, and therefore can be distinguished from biological tissues and three-dimensional tissues produced by other methods that do not use a fragmented extracellular matrix, depending on the presence or absence of the fragmented extracellular matrix.

[0017] In this embodiment, the term "adipocyte" includes not only differentiated cells such as mature adipocytes, but also undifferentiated cells such as adipose stem cells. For example, cells harvested from subcutaneous adipose tissue, epicardium-derived adipose tissue, etc. may be used as adipocytes, or the harvested cells may be induced to differentiate and used. Although there is no particular limitation on the adipocytes, when the adipose tissue constructed from the adipocytes is ultimately used as a tissue of a specific part of the living body, it is preferable to use those derived from the tissue corresponding to the tissue of the part. Examples of animal species from which the adipocytes are derived include humans, mice, rats, pigs, etc. Preferred adipocytes include mature adipocytes or adipose stem cells.

[0018] The term "extracellular matrix" refers to a substance present outside cells in an organism, and specific examples include collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, etc. The extracellular matrix in this embodiment is preferably a substance present outside animal cells, i.e., an animal extracellular matrix, more preferably contains collagen or elastin, even more preferably is collagen or elastin, and particularly preferably is collagen.

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

[0020] Conventional three-dimensional tissues have a low concentration of extracellular matrix such as collagen and a high cell density. Therefore, there are problems such as the three-dimensional tissue shrinking due to the traction force of cells during or after culture, and the three-dimensional tissue easily decomposing due to enzymes produced by cells during or after culture. In addition, for example, porous high-density collagen is also commercially available, but these cannot uniformly adhere collagen to cells, and it is difficult to collect cells for subsequent cell characteristic evaluation. The adipose tissue made of the three-dimensional tissue according to this embodiment has a higher content of extracellular matrix than that of conventional three-dimensional tissues, and is stable and unlikely to shrink.

[0021] The content of the extracellular matrix in the three-dimensional tissue may be 0.01 to 90% by weight, preferably 10 to 90% by weight, 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, the "extracellular matrix in the three-dimensional tissue" means the extracellular matrix constituting the three-dimensional tissue, and may be an endogenous extracellular matrix or an exogenous extracellular matrix. The "extracellular matrix in the three-dimensional tissue" also includes a fragmented extracellular matrix, which will be described later. That is, when the three-dimensional tissue contains an endogenous extracellular matrix, the concentration of the extracellular matrix constituting the three-dimensional tissue means 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 an extracellular matrix produced by extracellular matrix-producing cells, and "endogenous collagen" refers to collagen produced by collagen-producing cells that constitute a three-dimensional tissue. Endogenous collagen may be either fibrous collagen or non-fibrous collagen.

[0023] The term "exogenous extracellular matrix" refers to an extracellular matrix supplied from the outside. The artificial adipose tissue according to the present embodiment is composed of a three-dimensional tissue, and the three-dimensional tissue contains a 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 cows. The exogenous extracellular matrix may also be an artificial extracellular matrix. The fragmented extracellular matrix is ​​an exogenous extracellular matrix. When the extracellular matrix is ​​collagen, it is also called "exogenous collagen", which means collagen supplied from the outside, and specifically includes fibrous collagen, non-fibrous collagen, and the like. The exogenous collagen is preferably fibrous collagen. Examples of the fibrous collagen include type I collagen, type II collagen, and type III collagen, and preferably type I collagen. The fibrous collagen may be a commercially available collagen, and a specific example of the fibrous collagen is a freeze-dried type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. Exogenous non-fibrillar collagens include, for example, type IV collagen.

[0024] In an exogenous extracellular matrix, the animal species of origin may be different from that of the cells, and in cases where the cells include extracellular matrix-producing cells, the animal species of origin may be different from that of the extracellular matrix-producing cells, i.e., the exogenous extracellular matrix may be a xenogeneic extracellular matrix.

[0025] The three-dimensional tissue includes a fragmented extracellular matrix. The "fragmented extracellular matrix" means a fragmented extracellular matrix such as collagen. The extracellular matrix from which the fragmented extracellular matrix is ​​derived may be one type, or multiple types of extracellular matrices may be used in combination. Conventionally, extracellular matrices such as collagen have been dissolved in acidic aqueous solutions, but the concentration was about 0.1 to 0.3% by weight, and it was not possible to dissolve a large amount of them. Therefore, it was difficult to increase the amount of extracellular matrix such as collagen in the three-dimensional tissue by the conventional method. The fragmented extracellular matrix according to this embodiment is hardly soluble in water, but it is presumed that by dispersing it in an aqueous medium described later, it becomes easier to contact cells including fat cells 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" means collagen such as fibrous collagen that has been fragmented and that maintains 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] The method for fragmenting the extracellular matrix such as collagen is not particularly limited, and the extracellular matrix may be fragmented using a homogenizer such as an ultrasonic homogenizer, an agitation homogenizer, or a high-pressure homogenizer. When using an agitation homogenizer, the extracellular matrix may be homogenized as it is, or may be homogenized in an aqueous medium such as physiological saline. It is also possible to obtain millimeter-sized or nanometer-sized fragmented extracellular matrix by adjusting the homogenization time, number of times, etc.

[0028] The diameter and length of the fragmented extracellular matrix can be determined by analyzing individual fragmented extracellular matrix by electron microscopy.

[0029] According to the production method of this embodiment, it is possible to produce artificial adipose tissue consisting of a large-sized three-dimensional tissue structure with a thickness of 1 mm or more using a relatively small number of cells.

[0030] The artificial adipose tissue made 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 that of living tissue, and is suitable as a substitute for laboratory animals and a transplant material.

[0031] Here, "thickness of a three-dimensional structure" means the distance between both ends in a direction perpendicular to the main surface when the three-dimensional structure is sheet-shaped or rectangular. When the main surface is uneven, the thickness means the distance at the thinnest part of the main surface. When the three-dimensional structure is spherical, the thickness means its diameter. Furthermore, when the three-dimensional structure is ellipsoidal, the thickness means its minor axis. When the three-dimensional structure is approximately spherical or ellipsoidal and has an uneven surface, the thickness means the shortest distance between the two points where a straight line passing through the center of gravity of the three-dimensional structure intersects with the surface.

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

[0033] The size of lipid droplets can be used as an index of the maturity of cultured fat cells. Lipid droplets are intracellular organelles that store lipids such as triglycerides (neutral fats) and cholesterol, and have a droplet-like shape because the lipids are covered with a single membrane of phospholipids. In addition, expression of proteins specific to adipose tissue (perilipin, etc.) is observed on the surface of the phospholipids. The size of lipid droplets in mature fat cells varies, but when the average lipid droplet size is 20 μm or more, for example, the fat cells can be considered to be somewhat mature, that is, mature fat cells.

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

[0035] In addition, the expression profile of gene markers such as adipogenesis markers and lipolysis markers can be used as an index of maturity of cultured adipocytes. Examples of adipogenesis 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 stage markers of adipogenesis, and GLUT-4 can be used as a late stage marker of adipogenesis. 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. It has been reported that during adipogenesis, PPARγ2 expression increases, followed by a steady or decreased state (B. Galateanu et al., Int. J. Mol. Sci. 2012 (13) 15881-15900 and A. Soukas et al., J. Biol. Chem. 2001 (276) 34167-34174). Thus, adipogenesis is considered to be more advanced in tissues at a constant PPARγ2 expression stage compared to tissues at a stage where PPARγ2 is increased. FABP4 is another early marker required for transporting fatty acids to the membrane for efflux. Thus, for example, tissues showing a more typical linear increase in FABP4 expression profile are considered to be at a more early 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. In tissues undergoing early stages of adipogenic differentiation, GLUT-4 expression is weak (S.-W. Qian et al., BMC Dev. Biol. 2010 (10) 47). Thus, for example, tissues showing an earlier increase in GLUT-4 expression are more likely to be undergoing adipogenic differentiation.

[0038] The artificial adipose tissue of the present embodiment may contain cells other than fat cells, and may contain extracellular matrix-producing cells. "Extracellular matrix-producing cells" refers to cells that secrete extracellular matrix such as collagen. In other words, the artificial adipose tissue may contain endogenous extracellular matrix. Examples of the extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, and are preferably fibroblasts. Examples of preferred fibroblasts include human dermal fibroblasts (NHDF), human cardiac fibroblasts (NHCF), and human gingival fibroblasts (HGF). Examples of other cells other than adipocytes include vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVEC)), colon cancer cells (e.g., human colon 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, adhesive cells (e.g., immune cells), smooth muscle cells (e.g., aortic smooth muscle cells (Arota-SMC)), keratinocytes (e.g., human epidermal keratinocytes), and the like.

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

[0040] (artificial skin) The artificial skin according to the present embodiment includes a plurality of layers, one of which is a layer made of the above-mentioned artificial adipose tissue. That is, the artificial skin according to the present embodiment includes a first layer and a second layer, and includes the above-mentioned artificial adipose tissue as the first layer. The artificial skin may 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 may include more layers. The order of the layers does not matter, but it is preferable that the first layer, the second layer, and the third layer are laminated in this order. However, even in this case, the first layer, the second layer, and the third layer may be in this order, and there is no restriction on including another layer, for example, below the first layer, between the first layer and the second layer, between the second layer and the third layer, or above the third layer.

[0041] Each layer of the artificial skin according to this embodiment can be distinguished based on the type of cells contained therein, differences in cell distribution, etc. For example, by dyeing the artificial skin with a different color for each type of cell, each layer is shown in a different color, and each layer can be distinguished by a cross-sectional photograph, etc. Also, for example, by dyeing all the cells contained in the artificial skin, each layer can be distinguished by the difference in the density of the stained cells in a cross-sectional photograph, etc.

[0042] The layers other than the first layer (such as the second layer, the third layer, the fourth layer, and the fifth layer) contain cells. The cells may contain the above-mentioned adipocytes, or may contain cells other than adipocytes. The second layer preferably contains fibroblasts or keratinocytes, more preferably contains fibroblasts, and even more preferably contains human skin-derived fibroblasts or human cardiac fibroblasts. The third layer preferably contains fibroblasts or keratinocytes, more preferably contains keratinocytes, and even more preferably contains 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 is because by stacking in this way, an artificial skin closer to the skin structure in the human body can be obtained.

[0043] In addition, layers other than the first layer (such as the second layer, the third layer, the fourth layer, and the fifth layer) may contain an extracellular matrix or may contain a fragmented extracellular matrix. It is preferable that the second layer contains a fragmented extracellular matrix. The same extracellular matrix and fragmented extracellular matrix as those described above can be used. By including a fragmented extracellular matrix, a layer with appropriate spacing between cells can be formed.

[0044] The artificial skin according to the present embodiment has a thickness of preferably 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 an artificial skin has a structure closer to that of living tissue, and is suitable as a substitute for laboratory animals and as a transplant material.

[0045] "Thickness of artificial skin" means the distance from the surface of the topmost tissue to the bottom surface of the bottommost tissue. For example, in the case of a three-layered tissue including epithelial tissue, dermis tissue, and subcutaneous tissue, it means the distance from the surface of the epithelial tissue to the bottom surface of the bottommost subcutaneous tissue (total thickness of the three-layered tissue).

[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 of this embodiment is made of the above-mentioned artificial adipose tissue. In the artificial adipose tissue, the average size of the fat droplets of the fat cells is preferably 20 μm to 180 μm, more preferably 100 μm to 180 μm. The size of the fat droplets may be 250 μm or less, 200 μm or less, 180 μm or less, or 150 μm or less. The size of the fat droplets may be 15 μm or more, 30 μm or more, 50 μm or more, or 80 μm or more. The size of the fat droplets may be a value on the 7th day, 10th day, 14th day, or 21st day of culture.

[0048] The artificial skin can be used as a substitute for the skin of a living body, as a substitute for an experimental animal, as a transplant material, etc. Specifically, the artificial skin can be used in tests for evaluating the skin permeability, skin safety, skin corrosivity, skin irritation, etc. of a compound, in cosmetic assay screening, in pharmaceutical screening, in a pathological in vitro model, and as a transplant material for orthopedic surgery or burn surgery.

[0049] (Method of manufacturing artificial adipose tissue) The method for producing artificial adipose tissue according to this embodiment includes the steps of: (1) contacting cells, including adipocytes, with fragmented extracellular matrix in an aqueous medium (hereinafter also referred to as step (1)); and (2) the step of culturing the cells in contact with the fragmented extracellular matrix (hereinafter also referred to as step (2)).

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

[0051] In the present embodiment, the "cells including adipocytes" in step (1) may include cells other than adipocytes, or may include extracellular matrix-producing cells. The "extracellular matrix-producing cells" refer to cells that secrete extracellular matrix such as collagen. Examples of the extracellular matrix-producing cells include mesenchymal cells such as fibroblasts, chondrocytes, and osteoblasts, and preferably fibroblasts. Examples of preferred fibroblasts include human dermal fibroblasts (NHDFs), human cardiac fibroblasts (NHCFs), and human gingival fibroblasts (HGFs). Examples of other cells other than adipocytes include vascular endothelial cells (e.g., human umbilical vein-derived vascular endothelial cells (HUVEC)), colon cancer cells (e.g., human colon 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, adhesive cells (e.g., immune cells), smooth muscle cells (e.g., aortic smooth muscle cells (Arota-SMC)), keratinocytes (e.g., human epidermal keratinocytes), and the like.

[0052] "Aqueous medium" means a liquid containing water as an essential component. There is no particular limitation on the aqueous medium, so long as the fragmented collagen and cells can exist stably. Examples of the aqueous medium include physiological saline such as phosphate buffered saline (PBS), Dulbecco's Modified Eagle medium (DMEM), and liquid medium for vascular endothelial cells (EGM2). The liquid medium may be a mixed medium in which two types of media are mixed. From the viewpoint of reducing the load on the cells, the aqueous medium is preferably a liquid medium.

[0053] There is no particular limitation on the method for contacting the fragmented extracellular matrix with cells, including adipocytes, in an aqueous medium, for example, a method of adding a dispersion of the fragmented extracellular matrix to a culture medium containing cells, a method of adding cells to a medium dispersion of the fragmented extracellular matrix, or a method of adding the fragmented extracellular matrix and cells to a previously prepared aqueous medium.

[0054] The concentration of the fragmented extracellular matrix in the aqueous medium in step (1) can be appropriately determined depending on the shape and thickness of the artificial adipose tissue consisting of the desired three-dimensional tissue, the size of the culture vessel, etc. For example, the concentration of the 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 5 The amount may be 0.1 to 100 mg, or 1 to 50 mg, relative to the number of cells.

[0056] The mass ratio of the fragmented extracellular matrix to the cells in step (1) 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:other cells in step (1) may be 99:1 to 9:1, or 80:20 to 50:50.

[0058] Between steps (1) and (2), a step of precipitating cells including adipocytes together with the fragmented extracellular matrix in the aqueous medium may be further included. By carrying out such a step, the distribution of the fragmented extracellular matrix and cells in the artificial adipose tissue consisting of a three-dimensional tissue becomes more uniform. The specific method is not particularly limited, and an example thereof includes a method of centrifuging a culture solution containing the fragmented extracellular matrix and cells.

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

[0060] In this embodiment, the method for producing a three-dimensional tissue may further include, after step (2), a step (3) of contacting cells and culturing the cells. The cells may be the same as or different from the cells used in step (1). For example, when 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, when 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, and both the cells used in step (1) and the cells used in step (3) may include cells other than extracellular matrix-producing cells. By the above step (3), an artificial adipose tissue consisting of a three-dimensional tissue with a two-layer structure can be produced. By a similar method, an artificial skin including multiple layers can be produced. The method for producing the artificial skin will be described later.

[0061] In step (2), the method of culturing the cells in contact with the fragmented extracellular matrix is ​​not particularly limited, and can be performed by 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 medium may be 6 to 8, or 7.2 to 7.4. The production method according to this embodiment does not require a culture medium with a complex composition used in conventional two-dimensional culture and three-dimensional tissue production, and a medium that can be easily prepared such as DMEM can be used. The medium is not particularly limited, and a suitable medium can be selected depending on the type of cells to be cultured. Examples of the medium include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The medium may be a medium containing serum or a serum-free medium. The medium may be a mixed medium in which two types of media are mixed. In addition, according to the production method according to this embodiment, a sufficiently differentiated artificial adipose tissue can be obtained in a much shorter culture time than in 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 only indicates the time required to obtain sufficiently differentiated adipose tissue, and does not prevent further culture.

[0062] The cell density in the medium in step (2) 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 step (2) can be set to 1 to 10 8 cells / mL, 3 ~10 7 The cell density in the 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 produced by the production method according to the present embodiment preferably has a contraction rate during culture of 20% or less, more preferably 15% or less, and even more preferably 10% or less. The contraction 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 on the first day after culture, and L3 represents the length of the corresponding part in the three-dimensional tissue on the third day after culture. Shrinkage rate (%) = {(L1-L3) / L1} x 100 In the above example, the contraction rate is calculated from the artificial adipose tissue on the first day after culture and the artificial adipose tissue on the third day after culture, but it may be calculated from the artificial adipose tissue at any time during the culture period, including the end of the culture. For example, it may be calculated from the artificial adipose tissue on the first day after culture and the artificial adipose tissue on the second day after culture, or it may be calculated from the artificial adipose tissue on the first day after culture and the artificial adipose tissue on the fifth day after culture, or it may be calculated from the artificial adipose tissue on the first day after culture and the artificial adipose tissue on the eighth day after culture.

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

[0065] The artificial adipose tissue consisting of a three-dimensional tissue produced by the production method according to the present embodiment has a survival rate of preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more after collagenase treatment at a collagenase concentration of 0.25%, at a temperature of 37° C., at pH 7.4, and for a reaction time of 15 minutes. Such a three-dimensional tissue is stable and unlikely to be decomposed by enzymes during or after culture.

[0066] Moreover, the manufacturing method according to the present embodiment can produce an artificial adipose tissue consisting of a three-dimensional structure that is stable and has cells distributed uniformly. Furthermore, the artificial adipose tissue produced by the manufacturing method according to the present embodiment maintains mature adipocytes for a longer period of time compared to conventional two-dimensional culture. Furthermore, the manufacturing method according to the present embodiment can produce an artificial adipose tissue consisting of a large-sized three-dimensional structure with a thickness of 1 mm or more using a relatively small number of cells.

[0067] (Manufacturing method of artificial skin) The method for producing artificial skin according to this embodiment includes the steps of: (1) contacting a first cell, including an adipocyte, with a first fragmented extracellular matrix in an aqueous medium (hereinafter also referred to as step (1)'); and (2) forming a first layer, the step including culturing the first cells in contact with the first fragmented extracellular matrix (hereinafter also referred to as step (2)'); (3) a step of forming a second layer, which includes contacting second cells with the first layer and culturing the second cells (hereinafter, also referred to as step (3)'); Includes.

[0068] The steps (1)' and (2)' may be the same as those described above in (Method for producing artificial adipose tissue). The aqueous medium, the first cells including adipocytes, the second cells, and the first fragmented extracellular matrix may be the same as those described above in terms of the aqueous medium, the cells including adipocytes, and the fragmented extracellular matrix.

[0069] The method for forming the first layer in step (2)' is not particularly limited, but when the thickness of the cultured cells reaches a desired thickness, the layer can be considered to have been formed. It is not necessary to start step (3)' after the formation of the first layer in step (2)' is completed, and 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 production method of this embodiment, a sufficiently differentiated adipose tissue (first layer) can be obtained in a much shorter culture time than 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 a sufficiently differentiated adipose tissue, and does not prevent further culture.

[0072] The second cells may be the same as or different from the first cells. In addition, the method for contacting and culturing the cells may be the same as the method described above (method for producing an artificial adipose tissue).

[0073] The second cells may be the same as those described above (artificial adipose tissue), but it is preferable that the second cells contain fibroblasts. By making the second layer a layer in which fibroblasts are differentiated, it is possible to obtain a structure closer to that of living skin.

[0074] Also, it is preferable that step (3)' is a step including contacting the second cells with a fragmented second extracellular matrix on the first layer in an aqueous medium, and culturing the second cells. Examples of the second extracellular matrix include those described above in (artificial adipose tissue). By including a fragmented extracellular matrix, a layer with appropriate spacing between cells can be formed. It is preferable that the second extracellular matrix contains collagen, and it is more preferable that the second extracellular matrix is ​​collagen.

[0075] The method for manufacturing artificial skin according to this embodiment may further include a step of forming a third layer, which includes contacting third cells with the second layer and culturing the third cells.

[0076] The cells used as the third cells include those described above (artificial adipose tissue), but it is preferable that the third cells include keratinocytes. By making the third layer a layer in which keratinocytes are differentiated, it is possible to obtain a structure closer to that of the skin of a living body.

[0077] According to the method for producing artificial skin in this embodiment, similar to that described above (artificial skin), 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.

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

[0079] (Adipocyte culture agent) The culture agent for adipocytes according to the present embodiment includes a fragmented extracellular matrix. In the culture agent for adipocytes according to the present embodiment, the fragmented extracellular matrix may have an average length of 100 nm to 200 μm, and an average diameter of the fragmented extracellular matrix may be 50 nm to 30 μm. In addition, the length of the fragmented extracellular matrix may be in the range of 100 nm to 200 μm for 95% of the entire fragmented extracellular matrix. In addition, the diameter of the fragmented extracellular matrix may be in the range of 50 nm to 30 μm for 95% of the entire fragmented extracellular matrix.

[0080] The term "culture agent for adipocytes" refers to a reagent for culturing adipocytes. The culture agent for adipocytes may be in a powder state or in a dispersion state in which a fragmented extracellular matrix is ​​dispersed in an aqueous medium. The type, size, manufacturing method, and method of using the culture agent for adipocytes may be the same as those shown in the above (artificial adipose tissue) and (method of manufacturing artificial adipose tissue). By including a fragmented extracellular matrix, differentiation of adipocytes is promoted. Therefore, the culture agent for adipocytes can also be regarded as a differentiation promoter. Furthermore, by including a fragmented extracellular matrix, dedifferentiation of mature adipocytes is less likely to occur. Therefore, the culture agent for adipocytes can also be regarded as a dedifferentiation inhibitor (a maintenance agent for mature adipocytes). Furthermore, according to the culture agent of this embodiment, even when a tissue having a thickness of, for example, 1 mm or more is formed, it is possible to form a tissue with little tissue shrinkage due to long-term culture, so that it is possible to form a tissue with a stable morphology for a long period of time while exerting a differentiation promotion effect and a dedifferentiation inhibitory effect.

[0081] (Method for evaluating the skin permeability of a compound) The method for evaluating the skin permeability of a compound according to this embodiment includes the following steps: Providing an artificial skin; contacting a test compound with an artificial skin; measuring the skin permeability of the test compound using an artificial skin contacted with the test compound; comparing the skin permeability of the test compound with a reference value; The artificial skin used is the artificial skin according to the present embodiment described above in (Artificial Skin) or the artificial skin produced by the production method according to the present embodiment described above in (Production Method of Artificial Skin). As described above, the artificial skin has a structure and thickness similar to that of the skin of a living body, and therefore can be used in skin permeability tests of compounds as a substitute for the skin of a living body.

[0082] The test compound is not particularly limited, and any compound that is usually used in a skin permeability test can be used.

[0083] The method for contacting the test compound with the artificial skin is not particularly limited, but if the test compound is a liquid, it can be, for example, directly applied or sprayed onto the surface of the artificial skin, and if the test compound is a solid, it can be dissolved in a solvent and then similarly applied or sprayed onto the surface of the artificial skin.

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

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

[0086] The present invention will be described in more detail and specifically below by showing examples, but the scope of the present invention is not limited to these examples. EXAMPLES

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

[0088] A freeze-dried type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. was dispersed in 10x phosphate buffered saline (x10 PBS) and homogenized for 5 minutes using a homogenizer to obtain fragmented collagen with a diameter of about 4.4 μm and a length of about 22.5 μm. The obtained fragmented collagen was washed with serum-free medium (DMEM) to obtain a medium dispersion of the fragmented collagen.

[0089] The fragmented collagen was dispersed in serum-containing 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 × 10 6The cells were mixed with primary mature mouse adipocytes in a 24-well plate transwell (IWAKI). 24 hours after seeding, the transwell was placed in a 6-well plate (IWAKI) containing 6 mL of medium (DMEM medium, Nacalai Tesque). The mature adipocytes were cultured for 14 days with medium changes every 4 days. Since the mixture containing cells and fragmented collagen was simply piled up at the start of culture and not centrifuged, the thickness in the direction of its own weight decreased and stabilized over the course of culture, and a three-dimensional tissue with a thickness of about 2 mm was obtained after 14 days of culture. The artificial adipose tissue consisting of the obtained three-dimensional tissue was stained with hematoxylin and eosin (HE) and / or immunostained with anti-perilipin antibody and histological evaluation was performed. In addition, lipids were stained with Nile red. The viability was evaluated using a Live / Dead Kit (Thermo Fisher Scientific), and the measurement of live / dead and analysis of fat vesicles was performed using ImageJ software (National Institutes of Health, USA). In addition, the diameters of 100 fat vesicles on the 7th and 14th days of culture were measured using an electron microscope and compared with those of fat cells in artificial adipose tissue produced by conventional two-dimensional culture.

[0090] The conventional method for producing artificial adipose tissue by two-dimensional culture was as follows. 3 × 10 5 Primary mature mouse adipocytes (cells / well) were seeded and cultured. Culture was continued in 2 mL of 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 the lipid droplets, a commercially available cover glass was floated on the medium, and the cells were attached and cultured underneath it.

[0091] HE staining showed that there was dense fragmented collagen in the three-dimensional tissue surrounding the mature adipocytes (Fig. 2A, arrows).The mature adipocytes showed a typical round ocelli shape with small cytoplasm and nuclei (Fig. 2A).

[0092] Lipid staining with Nile red confirmed lipid droplets in the cytoplasm. Single-locular shaped lipid droplets, which indicate mature adipocytes, were confirmed, indicating homogeneous and high refractionation of mature adipocytes in the 3D tissue (Figure 2B). Even when cultured for a long period of more than 14 days, the artificial adipose tissue maintained mature adipocytes well with a high viability, from 98.5% on day 2 to 94.6% on day 14 (Figure 2C, gray indicates live cells, white indicates dead cells). Immunostaining with anti-perilipin antibody confirmed that mature adipocytes in the 3D tissue on day 14 of culture maintained a large single-locular shape (54 ± 8 μm lipid vesicles on day 0) compared with adipocytes in the artificial adipose tissue produced by conventional 2D culture (Figure 3). On the other hand, in the artificial adipose tissue produced by conventional two-dimensional culture, mature adipocytes dedifferentiated into fibroblast-like cell morphology and showed significantly smaller vesicles by 3.7 times compared to mature adipocytes in the three-dimensional tissue on the 14th day of culture (Figure 4, n>100 fat vesicles counted, t-test). In addition, the artificial adipose tissue made of the three-dimensional tissue containing fragmented collagen maintained the size of fat vesicles from the 7th to the 14th day, whereas the artificial adipose tissue produced by conventional two-dimensional culture decreased in fat vesicle size from the 7th to the 14th day. This indicates that the three-dimensional tissue containing fragmented collagen takes a longer time to dedifferentiate compared to the artificial adipose tissue produced by conventional two-dimensional culture, and therefore can be maintained for a long period of time, and the culture time of mature adipocytes can be extended for 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 produced as follows, as shown in the schematic diagram in Figure 1.

[0094] A freeze-dried type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. was dispersed in 10x phosphate buffered saline (x10 PBS) and homogenized for 5 minutes using a homogenizer to obtain fragmented collagen with a diameter of about 4.4 μm and a length of about 22.5 μm. The obtained fragmented collagen was washed with serum-free medium (DMEM) to obtain a medium dispersion of the fragmented collagen.

[0095] Fragmented collagen was dispersed in serum-containing medium (DMEM) to a concentration of 10 mg / mL. 300 μL of the resulting dispersion (equivalent to approximately 3 mg of fragmented collagen) and 5 × 10 5 Human adipose stem cells from the 19-well insert (ACEA Bioscience) were mixed with the human adipose stem cells. 24 hours after seeding, 200 μL of medium (DMEM medium, Nacalai Tesque) was added. The medium was replaced every 2 days and cultured for 19 days. Of the 19 days, 2 days correspond to the proliferation period, and the following 17 days correspond to the differentiation period. Since the mixture containing cells and fragmented collagen was simply piled up at the start of culture and was not centrifuged, the thickness in the direction of its own weight decreased and stabilized over the course of culture, and a three-dimensional tissue with a thickness of about 2 mm was obtained after 14 days of culture. The artificial adipose tissue consisting of the three-dimensional tissue on the 17th day of differentiation (19th day of culture) was stained with hematoxylin and eosin (HE) and / or immunostained with anti-perilipin antibody, and histological evaluation was performed.

[0096] HE staining showed the presence of high density fragmented collagen in the 3D tissue (arrow in Fig. 5). Large round, unilocular lipid droplets, which indicate mature adipocytes, were also identified (enlarged view in Fig. 5). Some mature adipocytes were larger than 100 μm.

[0097] The conventional method for producing artificial adipose tissue by two-dimensional culture was as follows. 1 × 10 4 Human adipose stem cells (cells / well) were seeded and cultured. After seeding, the cells were cultured in 500 μL of D-MEM for two days to allow proliferation, and then the medium was replaced with a medium for promoting differentiation. The medium for promoting differentiation was D-MEM with the addition of PGM-2 Singlequot Kit PT-9502 (manufactured by LONZA), which contains growth supplements (indomethacin, 3-isobutyl-1-methylxanthine, dexamethasone, and insulin). The cells were cultured in 500 μL, and the medium was replaced once a week.

[0098] In the artificial adipose tissue produced by conventional two-dimensional culture, lipid vesicles in a droplet state were found scattered even on the 37th day of differentiation (Figure 6). Furthermore, when confirmed by lipid staining with Nile red, only small lipid droplets were confirmed, and no mature fat cells larger than 100 μm were confirmed, as in the three-dimensional tissue containing fragmented collagen.

[0099] Furthermore, in the artificial adipose tissue produced by conventional two-dimensional culture, lipid vesicles in a droplet state were still found even on the 72nd day of differentiation, and not all fat cells had differentiated (Figure 6). As fat increased, the fat cells separated and floated in the culture medium, making it difficult to replace the culture medium.

[0100] Immunostaining with anti-perilipin antibody confirmed that the adipose stem cells in the 3D tissue on the 7th day of culture formed large monocular shapes (fat vesicles of 4 μm ± 2 μm on the 7th day) compared with the adipose stem cells in the artificial adipose tissue produced by conventional 2D culture (Figure 7). On the other hand, the artificial adipose tissue produced by conventional 2D culture showed vesicles that were significantly twice as small as the adipocytes in the 3D tissue on the 7th day of culture (Figure 8, n>100 fat vesicles counted, t-test). In addition, the artificial adipose tissue made of the 3D tissue containing fragmented collagen had fat vesicles that more than doubled in size (10 μm ± 4 μm) from the 7th day to the 14th day, while the size of the fat vesicles in the artificial adipose tissue produced by conventional 2D culture was almost unchanged from the 7th day to the 14th day.

[0101] These results demonstrate that artificial adipose tissue consisting of a three-dimensional structure containing fragmented collagen undergoes faster differentiation 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 produced as follows, as shown in the schematic diagram of FIG.

[0103] A freeze-dried type I collagen derived from pig skin manufactured by Nippon Ham Co., Ltd. was dispersed in 10x phosphate buffered saline (x10 PBS) and homogenized for 5 minutes using a homogenizer to obtain fragmented collagen with a diameter of about 4.4 μm and a length of about 22.5 μm. The obtained fragmented collagen was washed with serum-free medium (DMEM) to obtain a medium dispersion of the fragmented collagen.

[0104] Fragmented collagen was dispersed in serum-containing 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 × 10 collagen in DMEM collected from the subcutaneous tissue of a rat. 6 The cells were mixed with mature rat adipocytes in a 24-well plate Transwell (IWAKI Co., Ltd.). The plate was placed in an incubator and cultured for 24 hours.

[0105] Next, 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 min. Then, 1 mL of the above-mentioned fragmented collagen medium dispersion (equivalent to approximately 10 mg of fragmented collagen) and 5×10 5 The cells were mixed with human dermal fibroblasts (NHDF) and 100 μL was distributed into 24-well inserts. The plate was placed in an incubator and cultured for 24 hours.

[0106] Next, the medium in the insert was aspirated on the collagen gel of the NHDF, and the insert was 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 on the collagen gel of the NHDF was aspirated, and 2×10 6Normal human epidermal keratinocytes (#KK-4009, 1 vial = 500,000 cells, manufactured by KURABO) were added to the insert. 1 mL of DMEM 5% FBS:EpiLife (#C-2517A, manufactured by Invitrogen) (1:1) medium was added to the outside of the insert, and 1 mL was added again to the outside of the insert after 1 hour.

[0107] The inner and outer medium was gently aspirated, and ascorbic acid was diluted 100-fold in DMEM 5% FBS:EpiLife (1:1) medium and added to the outer side of the insert as 500 μL of differentiation medium. No medium was added to the inner side of the insert. The medium outside the insert was changed every day until the 7th day of differentiation.

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

[0109] The artificial skin on the 7th day of keratinocyte differentiation produced as described above was stained with hematoxylin and eosin (HE) and histologically evaluated. The artificial skin had appropriate intercellular distances in all layers, including the lower layer containing fat cells (subcutaneous tissue), the middle layer containing fibroblasts (dermal tissue), and the upper layer containing keratinocytes (epidermal tissue), demonstrating that the structure was similar to that of skin tissue in vivo (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). In addition, the artificial skin on the 7th day of keratinocyte differentiation had a thickness (distance from the surface of the epithelial tissue to the bottom surface of the lowest subcutaneous tissue) of approximately 4.3 mm, demonstrating that the thickness was also similar to that of skin tissue in vivo.

[0110] 1×10 adipocytes for adipose tissue production 6 cells to 5 × 10 5When the amount of collagen fragmented used was changed from 10 mg to 15 mg, similar results were observed (Figures 12 and 13). The relationship between the differentiation time of keratinocytes and the thickness of the three-layered artificial skin is shown in Figure 14. It was shown that the decrease in skin thickness converges and stagnation occurs between days 3 and 7 of keratinocyte differentiation.

[0111] (Example 4: Promotion of adipogenesis of adipose stem cells in three-dimensional tissue) As in Example 2, the expression of adipogenic genes in three-dimensional tissue containing fragmented collagen produced using human adipose stem cells and in artificial adipose tissue 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 above three-dimensional tissue and the artificial adipose tissue produced by conventional two-dimensional culture using PureLink RNA Micro Kit (Invitrogen) according to the kit's protocol. The extracted RNA was quantified using Nanodrop (registered trademark) N1000 (Thermo Fisher Scientific). 1 μg of RNA was converted to cDNA using High Capacity RNA-to-cDNA Kit (Applied Biosystems) according to the kit's protocol. PCR was performed by amplifying 70 ng of cDNA with 0.3 μM forward and reverse primers using iTaq (registered trademark) Universal SYBR Green Supermix (BioRAD). The sequence of each primer used, the optimal amplification cycle number and temperature conditions for RT-PCR are shown in Table 1. cDNA synthesis and RT-qPCR reaction were performed using StepOnePlus (registered trademark) Real-time PCR System (Thermo Fisher Scientific).

[0113] [Table 1]

[0114] Adipogenesis switches from the proliferation stage to the differentiation stage, in which preadipocytes express the first early adipogenic genes (e.g., FABP4, PPARγ2) when they become immature adipocytes, and then express late genes (e.g., GLUT4) while accumulating lipids in fat vesicles. The expression of early and late markers was found to gradually increase throughout the culture period, but differences in expression were observed between the three-dimensional tissue and the artificial adipose tissue produced by two-dimensional culture. The amount of mRNA in the three-dimensional tissue containing the above-mentioned fragmented collagen tended to be higher than that of the artificial adipose tissue produced by two-dimensional culture, with the expression of the early gene FABP4 being up to 5.5 times higher on day 7, and the expression of the late gene GLUT4 being 8.3 times higher on day 21 (Figure 15).

[0115] The expression of the PPARγ2 gene continued to increase in the artificial adipose tissue produced by two-dimensional culture, but in the above three-dimensional tissue, the expression profile showed a slight increase from day 7 to day 21, but was almost constant. In three-dimensional tissue, PPARγ2 is already at a constant expression stage, and adipogenesis is considered to be more advanced. Another early marker, FABP4, was almost constant from day 7 to day 21 in the three-dimensional tissue. On the other hand, in the artificial adipose tissue produced by two-dimensional culture, increased expression was observed from day 7 to day 21. The expression of insulin-stimulated GLUT-4, a late marker, rapidly increased from day 14 in the three-dimensional tissue, while the expression of GLUT-4 in the artificial adipose tissue produced by two-dimensional culture was observed very weakly. These results showed that the above three-dimensional tissue showed gene expression 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 fragmented extracellular matrix being homogenized fragmented collagen.

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

3. The artificial adipose tissue according to claim 1 or 2, having a thickness of less than 200 μm to 10 mm.

4. The artificial adipose tissue described in any one of claims 1 to 3, wherein the average length of the fragmented extracellular matrix is ​​100 nm to 200 μm.

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

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

7. The artificial skin of claim 6 , wherein the second layer comprises fibroblasts.

8. The artificial skin of claim 6 or 7, further comprising a third layer, the third layer comprising keratinocytes.

9. The artificial skin according to any one of claims 6 to 8, having a thickness of more than 200 μm to 10 mm.

10. (1) 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 fragmented extracellular matrix is ​​homogenized fragmented collagen.

11. The method of claim 10 , wherein the extracellular matrix comprises collagen.

12. The method according to claim 10 or 11, wherein the average length of the fragmented extracellular matrix is ​​from 100 nm to 200 μm.

13. The method according to any one of claims 10 to 12, wherein the fragmented extracellular matrix is ​​dispersible in an aqueous medium.

14. (1) contacting first cells, including adipocytes, with a first fragmented extracellular matrix in an aqueous medium; and (2) forming a first layer comprising culturing the first cells in contact with the first fragmented extracellular matrix; (3) forming a second layer, the step further comprising contacting second cells with the first layer and culturing the second cells; Including, A method for producing artificial skin, wherein the fragmented extracellular matrix is ​​homogenized fragmented collagen.

15. The method for producing artificial skin according to claim 14, wherein the average length of the first fragmented extracellular matrix is ​​100 nm to 200 μm.

16. The method for producing artificial skin 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 described in any one of claims 14 to 16, wherein the second cells include fibroblasts.

18. forming a third layer, further comprising contacting the second layer with third cells and culturing the third cells; The method for producing artificial skin according to any one of claims 14 to 17, further comprising:

19. The method according to any one of claims 14 to 18, wherein the fragmented extracellular matrix is ​​dispersible in an aqueous medium.

20. An agent for promoting adipocyte differentiation, comprising a fragmented extracellular matrix, said fragmented extracellular matrix being homogenized fragmented collagen.

21. The differentiation promoting agent according to claim 20, wherein the fragmented extracellular matrix is ​​collagen having an average length of 100 nm to 200 μm.

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

Images