Skin model with controlled pigmentation, method for producing skin model, method of screening for agents to prevent, reduce, improve, or treat pigmentation spots, extracellular matrix protein gel for keratinocyte culture and method for producing the same

Abstract

To provide a skin model with controlled pigmentation.SOLUTION: A skin model with controlled pigmentation is provided by: discovering that melanin accumulation is affected by the irregular of a basement membrane; further discovering that melanin synthesis is affected by the hardness of an extracellular matrix protein gel imitating the basement membrane; and adjusting the hardness parameter and irregularity parameter of the extracellular matrix protein gel imitating the basement membrane.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a skin model with regulated pigmentation, a method for producing the same, and uses thereof. [Background technology]

[0002] Cell culture tests and animal tests have been used to analyze skin. Since skin is a complex and highly structured cell structure, cell culture tests alone are not sufficient for studying the interactions and cooperation between cells. In addition, animal tests using live animals are problematic from the perspective of animal welfare, so analysis using cultured skin models is being conducted as a new research method that does not use animals.

[0003] Skin models that mimic the structure of skin have become important tools in skin research. Skin models are used for various purposes, such as elucidating the physiological functions of skin, evaluating the performance of skin care products, and searching for active ingredients. In order to form an epidermal layer in a skin model, it is necessary to culture epidermal keratinocytes and then expose the stratum corneum side of the epidermal layer to a gas phase to induce differentiation to form the basal layer, spinous layer, granular layer, and stratum corneum. Skin models are broadly divided into epidermal models in which only the epidermal layer is formed by culturing keratinocytes, and skin models in which the epidermal layer is formed by culturing keratinocytes on the dermal layer formed by culturing dermal fibroblasts. In addition to keratinocytes and dermal fibroblasts, the cultured skin model can be prepared so that various cells contained in the epidermis or dermis, such as melanocytes and adipocytes in the subcutaneous fat layer under the dermis, are further arranged (Patent Document 1: JP 2010-193822 A, Patent Document 2: JP 2012-235921 A). Furthermore, in order to provide thickness to the cultured skin model, irregularities are formed in the base (Patent Document 3: WO 2017 / 222065 A).

[0004] Conventional skin models have been prepared by coating a flat plate or substrate with an extracellular matrix component that serves as a scaffold, seeding cells on the scaffold, and allowing them to proliferate, differentiate, and / or mature. Although the skin models prepared in this way mimicked the skin structure to some extent, they did not fully reproduce the unevenness just below the basement membrane. These uneven structures are usually of regular depth and spacing, and have been suggested to be important for the homeostasis of the epidermis, including the maintenance and proliferation of epidermal stem cells, and the normal differentiation of the stratum corneum to the barrier function (Non-Patent Document 1: Am J Physiol Cell Physiol. 2022 Dec 1; 323(6): C1807-C1822, Non-Patent Document 2: Development. 2020 Nov 15; 147(22): dev194100.).

[0005] On the other hand, the above-mentioned uneven structure is also present in the spot area and is called the papillary layer protuberance (rete ridge). The uneven structure in this spot is deeper than the normal uneven structure and the unevenness intervals are irregular, which is known as a lesion that characterizes spots. Furthermore, the hardness just below the basal layer is also considered to be important for epidermal homeostasis such as the maintenance of the basement membrane of epidermal cells, cell proliferation, and epidermal thickening. A skin model that mimics the hardness just below the basement membrane has also been constructed (Non-Patent Document 3: J Cell Sci. 2018 May 16;131(10):jcs215780, Non-Patent Document 4: Nature Aging volume 2, pages592-600 (2022)). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2010-193822 A [Patent Document 2] JP 2012-235921 A [Patent Document 3] International Publication No. 2017 / 222065 [Non-patent literature]

[0007] [Non-Patent Document 1] Am J Physiol Cell Physiol. 2022 Dec 1;323(6):C1807-C1822 [Non-Patent Document 2] Development. 2020 Nov 15;147(22):dev194100. [Non-Patent Document 3] J Cell Sci. 2018 May 16;131(10):jcs215780. [Non-Patent Document 4] Nature Aging volume 2, pages592-600 (2022) Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors predicted that the papillary layer elevation of pigmentation is involved in melanin accumulation in pigmentation sites and enhanced melanin synthesis ability of melanocytes, but the relationship and mechanism thereof remained unknown. Furthermore, a model has not yet been developed that examines and establishes the effects of the papillary layer structure on pigmentation and melanin production in age spots. In order to solve the above problems in pigmentation phenomena such as age spots, it is necessary to adjust both the parameters (depth, interval) and hardness (hardness) of the uneven structure just below the basement membrane in a skin model. Therefore, the present invention aims to provide a skin model in which the uneven depth, interval, and hardness are adjusted. [Means for solving the problem]

[0009] The present inventors came up with the idea that it is necessary to simulate the unevenness just below the basement membrane in a skin model with regulated pigmentation such as age spots, and prepared a skin model using an extracellular matrix protein gel having a predetermined uneven surface and gel hardness in manufacturing the skin model. They then found that in the skin model, the distribution of melanosomes provided to keratinocytes changes depending on the unevenness depth and unevenness interval (pitch) (hereinafter referred to as unevenness grain size) of the extracellular matrix protein gel simulating the area just below the basement membrane. Furthermore, they found that the lower the gel hardness, the more melanin synthesis is promoted.

[0010] Based on these new findings, by controlling the unevenness grain size and gel hardness of the extracellular matrix protein gel, it is possible to regulate differences in the distribution of melanosomes within keratinocytes and the amount of melanin produced, thereby providing a skin model with regulated pigmentation, which has led to the present invention. Thus, the present invention relates to: [1] A skin model with regulated pigmentation comprising an extracellular matrix protein gel having an uneven surface, keratinocytes seeded on the gel, and further melanosomes or seeded melanocytes. [2] The skin model described in item 1, wherein pigmentation is regulated by the localization of melanosomes taken up by keratinocytes around the nucleus and / or in the cytoplasm and around the cell membrane. [3] The skin model according to item 1 or 2, wherein the extracellular matrix protein gel having an uneven surface is a gel transferred from a mold having predetermined unevenness parameters. [4] The skin model according to Item 3, wherein the extracellular matrix protein gel having an uneven surface is produced by irradiating a protein aqueous solution or a physical gel with a quantum beam while a mold having predetermined unevenness parameters is in contact with the gel, hardening the gel, and peeling the gel off from the mold. [5] The skin model according to any one of items 1 to 4, wherein the pigmentation-adjusted skin model is one in which a roughness parameter of the rough surface of the extracellular matrix protein gel and a hardness parameter of the extracellular matrix protein gel are adjusted to correspond to a predetermined melanin distribution and / or melanin synthesis in a cell culture obtained by culturing the cells with the gel. [6] The skin model according to item 5, wherein the protein is prepared so that the extracellular matrix protein gel has unevenness parameters of an unevenness depth of 0 to 200 μm and an unevenness width of 0 to 1,500 μm. [7] The skin model according to item 5, wherein the unevenness parameters of the extracellular matrix protein gel are achieved on an uneven surface obtained by transferring the extracellular matrix protein gel to a mold having a surface roughness of sandpaper roughness (grain size) #12 to #2000 (compliant with JIS R 6010). [8] The skin model according to any one of items 1 to 7 for comparative studies between pigmented and non-pigmented areas. [9] The skin model according to any one of items 1 to 8, wherein the protein is prepared so that the hardness parameter of the extracellular matrix protein gel is 0.5 kPa to 200 kPa when measured by any one of a static test method, a lateral vibration method, an ultrasonic method, a rheometer, a nano- or micro-indentation method, or a coherence elastography (OCE).

[10] A method for producing a skin model, comprising: A step of laminating a mold having a surface roughness of sandpaper roughness (grain size) #12 to #2000 (based on JIS R 6010) on an extracellular matrix protein aqueous solution or physical gel to obtain a laminate; irradiating the laminate with a quantum beam of 5 to 50 kGy; removing the mold from the irradiated laminate to obtain an extracellular matrix protein gel having an uneven surface; a step of immersing the extracellular matrix protein gel in phosphate buffered saline (PBS), a medium, or the like, and incubating at 37°C to remove uncrosslinked components; seeding keratinocytes on the extracellular matrix protein gel and adding melanosomes or melanin or seeding melanocytes; Culturing the cells in a medium; and A process of culturing cells by exposing them to a gas phase The manufacturing method comprising the steps of:

[11] A skin model prepared by the manufacturing method described in item 10.

[12] A method for screening for an agent for preventing, reducing, improving or treating pigmentation, comprising: Culturing the skin model according to items 1 to 9 and 11 in a medium containing a candidate component; Identifying the location of melanin accumulation in keratinocytes; A step of comparing the melanin accumulation location in keratinocytes of a skin model cultured in a control medium containing no candidate component, and selecting a candidate component that causes a change in the accumulation location as an agent for preventing, reducing, improving or treating pigmentation. The screening method comprising the steps of:

[13] A method for screening for an agent for preventing, reducing, improving or treating pigmentation, comprising: Culturing the seeded melanocytes in the skin model according to items 1 to 9 and 11 in a medium containing a test drug; Identifying an indicator of melanin synthesis in melanocytes; A step of comparing the melanin synthesis index in melanocytes of a skin model cultured in a control medium containing no candidate component, and selecting a candidate component whose melanin synthesis index shows a decrease in melanin synthesis as an agent for preventing, reducing, improving or treating pigmentation. The screening method comprising the steps of:

[14] The screening method according to Item 13, wherein the melanin synthesis indicator is the amount of melanin.

[15] The screening method according to Item 13, wherein the melanin synthesis indicator is the protein amount or expression amount of a melanin synthesis-related gene.

[16] The screening method described in Item 17, wherein the melanin synthesis-related gene is selected from the group consisting of MLANA, PMEL.TYR, DCT, and TRP1.

[17] The screening method according to Item 13, wherein an increase in the number and length of dendrites in correlation with the melanin synthesis index is an indicator of the activation state of melanocytes as melanin producing cells.

[18] The screening method according to Item 13, wherein the activation state of melanocytes as melanin transport ability is indicated by an increase in the transfer of melanin from melanocytes to keratinocytes in correlation with the melanin synthesis index, and the indicator of melanin transport ability is the protein amount or expression amount of a melanin transport-related gene group consisting of MREG and MLPH.

[19] A method for producing an extracellular matrix protein gel for culturing keratinocytes, comprising: A step of laminating a mold having a surface roughness of sandpaper roughness (grain size) #12 to #2000 (based on JIS R 6010) on an extracellular matrix protein aqueous solution or physical gel to obtain a laminate; irradiating the laminate with a quantum beam of 5 to 50 kGy; removing the mold from the irradiated laminate to obtain an extracellular matrix protein gel having an uneven surface; The manufacturing method comprising the steps of: Effect of the Invention

[0011] By preparing a skin model using an extracellular matrix protein gel having a predetermined uneven surface and gel hardness, a skin model with regulated pigmentation can be obtained. In addition, by adjusting the gel hardness of the extracellular matrix protein gel, the amount of melanin production can be adjusted. [Brief description of the drawings]

[0012] [Figure 1]Figures 1 (A) and (B) are bright-field photographs showing the distribution of melanosomes taken up by keratinocytes. The distribution of melanosomes inside and outside keratinocytes (around the nucleus, around the cell membrane) is different in the skin model produced using the extracellular matrix protein gel transferred from a mold with a surface roughness of #600 (A) and the skin model produced using the extracellular matrix protein gel transferred from a mold with a surface roughness of #280 (B). Figure 1 (C) shows the results of a graph showing the distribution of melanosomes inside and outside keratinocytes (around the nucleus, around the cell membrane). [Diagram 2] Figure 2 shows bright-field photographs showing melanin production by melanocytes co-cultured with keratinocytes in a skin model produced using an extracellular matrix protein gel with a compressive modulus of 20 kPa (A) and a skin model produced using an extracellular matrix protein gel with a compressive modulus of 80 kPa (B). Figure 2 (C) shows the results of a graph showing the change in protein amount of melanin synthesis-related genes (MLANA and TRP1) in melanosomes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] One aspect of the present invention relates to a skin model with regulated pigmentation and a method for producing a skin model with regulated pigmentation. The skin model of the present invention is formed on an extracellular matrix protein gel gelled by a quantum beam. Another aspect of the present invention relates to a method for controlling pigmentation in a skin model using an extracellular matrix protein gelling technique, as well as an extracellular matrix protein gel used in the control method, and a method for producing the same. Note that in this specification, there are some descriptions using "~" as a description expression indicating a numerical range from a lower limit value to an upper limit value of a numerical value, but the numerical range in this description is a numerical range specified as being equal to or greater than the lower limit value and equal to or less than the upper limit value, including the lower limit value itself and the upper limit value itself.

[0014] [Skin model] In the present invention, the skin model with regulated pigmentation refers to a skin model with accumulated melanin. In one example, the skin model with regulated pigmentation refers to a skin model in which pigmentation, such as age spots, dullness, freckles, and uneven skin tone, is reproduced. The skin model with accumulated melanin refers to a skin model in which melanosomes containing melanin are taken up by keratinocytes, and includes any or all of distribution around the nucleus of keratinocytes, distribution in the cytoplasm, and distribution around the membrane of keratinocytes. The skin model with regulated pigmentation may simulate a site exhibiting pigmentation (age spots, dullness, uneven skin tone, and / or freckles) (hereinafter referred to as a pigmented site), or may simulate a site with normal skin color (hereinafter referred to as a non-pigmented site). The pigmented site can also be classified in more detail as a age spot site, a dull site, an uneven skin tone site, and a freckled site. The skin model with regulated pigmentation includes an extracellular matrix protein gel with an uneven surface that mimics the area just below the basement membrane, keratinocytes seeded on the gel, and melanosomes or seeded melanocytes. Melanocytes produce melanin during the culture process and provide it to keratinocytes as melanosomes, resulting in pigmentation. In addition, added melanosomes or purified melanin are also taken up by keratinocytes. The skin model simulating pigmented areas and the skin model simulating non-pigmented areas can each be used for skin color research (spots, dullness, uneven skin tone, and / or freckles).

[0015] The skin model of the present invention is also characterized in that the distribution of melanin can be controlled. The distribution of melanin can be controlled by adjusting the depth of the irregularities in the extracellular matrix protein gel that mimics the area just below the basement membrane. Furthermore, the skin model of the present invention is characterized in that the amount of melanin synthesis can be controlled. The amount of melanin synthesis can be controlled by adjusting the hardness of the extracellular matrix protein gel contained in the skin model.

[0016] Melanin is a brown to black pigment produced by melanocytes. Melanin is produced by tyrosine through the action of tyrosinase to produce dopa and dopaquinone, and is broadly divided into black eumelanin and yellow to brown pheomelanin. Melanosomes, organelles containing melanin, are donated to neighboring keratinocytes, particularly basal cells and spinous cells, and cause skin color, age spots, dullness, uneven skin tone, and freckles. Some of the melanosomes donated to keratinocytes migrate to the periphery of the nucleus to form a melanin cap, which protects the genome from ultraviolet rays. On the other hand, some melanosomes do not migrate to the periphery of the nucleus and are arranged around the cell membrane. The mechanism that controls the arrangement of melanosomes donated to keratinocytes has not yet been elucidated. It is possible to control the distribution of melanosomes donated to keratinocytes by adjusting the depth of the unevenness of an extracellular matrix protein gel that mimics the basement membrane. This allows control of the proportion of keratinocytes with melanosomes located near the membrane and the proportion of keratinocytes with melanosomes located near the nucleus, providing a method for modulating pigmentation in a skin model.

[0017] In one embodiment, a skin model is provided in which keratinocytes distributing melanosomes near the membrane are predominant. The term "keratinocytes distributing melanosomes near the membrane are predominant" refers to a case in which the total number of keratinocytes containing melanosomes and the number of keratinocytes distributing melanosomes near the membrane are counted, and the number of keratinocytes distributing melanosomes near the membrane is 50% or more, preferably 60% or more, and more preferably 80% or more. Such a skin model can be achieved by preparing a skin model with a depth of unevenness of an extracellular matrix protein gel simulating a basement membrane set to a sandpaper roughness (grain size) of #12 to #400.

[0018] In yet another embodiment, a skin model is provided in which keratinocytes distributing melanosomes around the nucleus are predominant. The term "keratinocytes distributing melanosomes around the nucleus are predominant" refers to a case in which the total number of keratinocytes containing melanosomes and the number of keratinocytes distributing melanosomes around the nucleus are counted, and the number of keratinocytes distributing melanosomes around the nucleus is 50% or more, preferably 60% or more, and more preferably 80% or more. Such a skin model can be achieved by preparing a skin model with a depth of unevenness of an extracellular matrix protein gel simulating a basement membrane set to a sandpaper roughness (grain size) of #400 to #2000.

[0019] The distribution of melanosomes may be measured using a commercially available optical microscope, or may be stained by immunohistochemistry and observed and measured using a fluorescent microscope, a confocal microscope, or a two-photon laser microscope, or may be observed and measured using an electron microscope, and is not particularly limited. The acquired image can be imported into computer analysis software using a standard method for image analysis and image analysis can be performed. In order to clarify the cells and nuclei, specific proteins or nucleic acids may be stained. When melanosomes are present in the vicinity of the nucleus (for example, a predetermined distance from the nucleus (within 3 μm, preferably within 1 μm)), it can be determined that the keratinocyte has melanosomes distributed around the nucleus, and when melanosomes are present in the vicinity of the cell membrane (for example, a predetermined distance from the cell membrane (within 3 μm, preferably within 1 μm)), it can be determined that the keratinocyte has melanosomes distributed near the membrane.

[0020] The cells used in the present invention may be primary keratinocytes obtained by finely chopping biological tissue, particularly skin tissue, and treating it with protease such as collagenase or trypsin, or primary keratinocytes propagated by subcultivation, or pluripotent stem cells such as ES cells, iPS cells, or Muse cells, or keratinocytes obtained by inducing differentiation from epidermal stem cells, or established keratinocytes. Preferably, primary cultured keratinocytes collected from biological tissue and seeded, or passaged keratinocytes obtained by further subculturing the primary cultured cells. Keratinocytes collected from biological tissue are likely to maintain the same properties as those in the living body, and are convenient for use in tests to investigate the efficacy and side effects of drugs, or in basic research.

[0021] The cells used in the present invention may include cells other than keratinocytes, for example, cells other than keratinocytes that constitute the epidermis (melanocytes, Langerhans cells, Merkel cells, etc.) and / or cells contained in dermal tissue (fibroblasts, nerve cells, mast cells, plasma cells, vascular endothelial cells, histiocytes, Meissner corpuscles, etc.). The skin model in the present invention may be an epidermis model composed of epidermis, or may be a skin model composed of dermis and epidermis.

[0022] [Extracellular matrix protein gel] Proteins constituting the extracellular matrix protein gel include collagen, laminin, elastin, fibrillin, fibronectin, entactin, polylysine, vitronectin, thrombospondin, tenascin, nidogen, proteoglycan, perlecan, etc., and the gel contains at least one of these proteins. From the viewpoint of reproducing the unevenness just below the basement membrane, it is preferable to use at least one selected from the group consisting of collagen, laminin, and entactin contained in the basement membrane. As the collagen, any collagen may be used, but it is preferable to use at least one selected from the group consisting of type I collagen, type III collagen, and type IV collagen, which are abundant in the basement membrane. In one embodiment, gelatin can be used as the extracellular matrix protein. The gelatin may be derived from any animal. The extracellular matrix protein gel functions as a scaffold for cell culture.

[0023] The extracellular matrix protein gel can be specified based on its concave-convex surface and gel hardness. The concave-convex surface of the extracellular matrix protein gel can be specified by a concave-convex parameter. Any parameter expressing concave-convexity can be used. As an example, it can be specified using the concave-convex depth, the concave-convex width, the concave-convex surface roughness, etc. As an example, the concave-convexity parameter can be set to an average concave-convex depth of 10 to 100 μm. From the viewpoint of reproducing or studying the papillary layer structure or the papillary layer protuberance in skin physiology, the concave-convexity depth is preferably 10 μm or more, more preferably 20 μm or more. From the viewpoint of reproducing or studying the papillary layer structure or the papillary layer protuberance in skin physiology, the concave-convexity depth is preferably 100 μm or less, more preferably 80 μm or less. In addition to or independently of the concave-convexity depth, the concave-convexity width can be set to an average concave-convexity width of 10 to 300 μm. From the viewpoint of reproducing or studying the papillary layer structure or papillary layer prominence from the viewpoint of skin physiology, the unevenness width is preferably 10 μm or more, more preferably 20 μm or more. From the viewpoint of reproducing and studying the papillary layer prominence in the pigmented area, the unevenness width is preferably 80 μm or less, more preferably 60 μm or less.

[0024] In one embodiment of the present invention, a concave-convex surface can be created on an extracellular matrix protein gel by transferring a mold having a concave-convex surface. The operation of preparing a processing object relative to a mold, hardening the processing object, and then removing the mold is called transfer. The transfer source and transfer destination have substantially equivalent concave-convex surface parameters. The transfer source mold can be prepared by transferring a resin such as polydimethylsiloxane (PDMS) to a member having a concave-convex surface, such as sandpaper, as a mold. Therefore, the concave-convex surface of the extracellular matrix protein gel created by transferring a mold having a concave-convex surface is equivalent to the concave-convex surface of a member having a concave-convex surface (such as sandpaper), and can be expressed by the concave-convex surface parameters of the member having a concave-convex surface. When sandpaper is used as the member having a concave-convex surface, the surface roughness of the sandpaper can be expressed by the grit size (#). The grit size of sandpaper usually corresponds to the grain size of abrasives for JISR6010 coated abrasives, and the unit "P" is used interchangeably in the JIS standard instead of "grain size (#)". In the present invention, the roughness of the original mold can be #180 (P180) to #1200 (P1200). From the viewpoint of studying the distribution of melanosomes or melanin, a roughness of 220 or more is preferable, more preferably 240 or more, and even more preferably 280 or more. From the viewpoint of studying melanin production ability, a roughness of 800 or less is preferable, and even more preferably 600 or less is preferable. The extracellular matrix protein gel transferred from the mold has unevenness corresponding to the unevenness of the original mold, and does not substantially contain other elements for forming such unevenness, such as beads or mesh. Therefore, it is clearly distinguished from the embodiment disclosed in Patent Document 3 (WO 2017 / 222065) in which the surface of a cell culture vessel having a convex portion on a porous membrane is coated with collagen or the like in that beads or mesh are not used as the convex portion.

[0025] The extracellular matrix protein gel can be specified by its hardness, and can be specified as a hardness of 0.5 kPa to 200 kPa when measured with a rheometer. From the viewpoint of maintaining an uneven surface, the gel hardness is preferably 10 kPa or more, more preferably 20 kPa or more. From the viewpoint of approximating the hardness of the dermis, it is preferably 100 kPa or less, and more preferably 80 kPa or less. The extracellular matrix protein gel can be hardened by any method, and as an example, the gel hardness can be adjusted by irradiating a predetermined amount of quantum beam to an aqueous solution containing a protein. Examples of quantum beams include gamma rays, electron beams, proton beams, heavy particle beams, X-rays, and synchrotron radiation. The dose of the quantum beam can be appropriately changed depending on the type of quantum beam used. As an example, when gamma rays are used, a cobalt-60 source or a cesium-137 source can be used, and when electron beams are used, a Cockcroft-Walton type or Van de Graaff type electron accelerator can be used. The quantum beam can be irradiated by using a tandem accelerator, a cyclotron accelerator, or the like.

[0026] By setting the gel hardness to 50 kPa to 100 kPa, the amount of melanin synthesis can be suppressed. From the viewpoint of suppressing the amount of melanin synthesis, 50 kPa or more is preferable, and 80 kPa or more is more preferable. From the viewpoint of not interfering with cell culture, 500 kPa or less is preferable, and 200 kPa or less is more preferable. Therefore, the skin model of the present invention may have an extracellular matrix protein gel hardness of 50 kPa to 200 kPa, particularly 50 to 100 kPa, and further 80 to 100 kPa. With such a skin model, it is possible to simulate skin before sunburn. By setting the gel hardness to 1 kPa to 40 kPa, it is possible to enhance the amount of melanin synthesis. From the viewpoint of maintaining the shape of the gel, 5 kPa or more is preferable, and 10 kPa or more is more preferable. From the viewpoint of promoting the amount of melanin synthesis, 40 kPa or less is preferable, and 20 kPa or less is more preferable. With such a skin model, it is possible to simulate skin after sunburn or middle-aged to elderly skin.

[0027] [Method of manufacturing extracellular matrix protein gel] The extracellular matrix protein gel according to the present invention can be produced by any method so as to have a predetermined unevenness parameter and gel hardness parameter. In order to have the predetermined unevenness parameter, it is preferable to produce the gel by transferring a mold having the predetermined unevenness parameter to an aqueous solution or physical gel of the extracellular matrix protein. The transfer is performed by attaching a mold having the predetermined unevenness parameter to an aqueous solution or physical gel of the extracellular matrix protein, performing a hardening treatment, and peeling off the mold. The hardening treatment may be irradiation of a quantum beam, addition of a polymerization initiator, heating treatment, and cooling treatment after heating. As an example, an aqueous solution containing 10% extracellular matrix protein is poured into a cell culture dish, a mold having a predetermined surface roughness is placed on the dish, and a quantum beam such as gamma rays or electron beams is irradiated to harden and gel the extracellular matrix protein without using chemicals. The mold is peeled off from the obtained extracellular matrix protein gel to produce an extracellular matrix protein gel having a predetermined unevenness parameter. The hardness of the gel can be adjusted by appropriately adjusting the hardening treatment.

[0028] [Method of manufacturing the skin model] The extracellular matrix protein gel produced according to the method for producing the extracellular matrix protein gel is placed in an air-liquid interface culture vessel, keratinocytes are seeded, and air-liquid interface culture is performed to produce the extracellular matrix protein gel. The air-liquid interface culture vessel is a cell culture vessel equipped with a cell culture insert for seeding cells. The bottom surface of the cell culture insert is made of a porous membrane, and by placing a culture medium in the cell culture vessel and stacking the cell culture insert, the cells seeded in the cell culture insert can contact the culture medium through the porous membrane on the bottom surface of the cell culture insert. On the other hand, when seeding cells in the cell culture insert, the inside of the cell culture insert is also filled with culture medium and cultured. By removing the culture medium in the cell culture insert after culture, the upper surface side of the proliferated cells comes into contact with air, which promotes the differentiation of the cultured keratinocytes to form a skin model having a basal layer, a spinous layer, a granular layer, and a stratum corneum. At this time, the culture medium in the cell culture vessel may be replaced with a culture medium that promotes differentiation. Air-liquid interface culture is also called three-dimensional culture.

[0029] In the present invention, the three-dimensional culture of keratinocytes can be performed by placing an extracellular matrix protein gel having a predetermined roughness parameter and a predetermined hardness on a cell culture insert and seeding keratinocytes on the extracellular matrix protein. Other configurations of the three-dimensional culture may be in accordance with known methods. For example, keratinocytes can be cultured at a density of 5×10 4 ~5×10 6The cells can be seeded at 100 cells / well. As the medium, for example, KG medium, EpilifeKG2 (Kurabo), Humedia-KG2 (Kurabo), Assay medium (TOYOBO), CnT-Prime, Epithelial culture medium (CELLnTEC), DMEM medium (GIBCO), or a medium in which 2-0-aD-glucopyranosyl-L-ascorbic acid-containing KGM and DMEM are mixed 1:1, can be used. After seeding, the keratinocytes are cultured and grown for 2 to 3 days at about 37°C in a humidified atmosphere of 5% CO2. The medium in the cell culture insert is then removed, the cells are exposed to air, and the cells are further cultured for 7 to 14 days at a temperature near the body temperature of the animal from which the cells were derived, for example, at 33 to 38°C, to induce differentiation of the keratinocytes, and a skin model having a basal layer, a spinous layer, a granular layer, and a stratum corneum can be obtained.

[0030] In producing a skin model, by appropriately selecting the roughness parameters and hardness parameters of the extracellular matrix protein gel, it is possible to produce a skin model that reproduces pigmentation, particularly spots, dullness, freckles, and / or uneven skin tone and skin color. Therefore, the production of a skin model in the present invention can also be said to be a method for controlling pigmentation in a skin model.

[0031] [Screening method] In yet another aspect, the present invention relates to a method for screening for a pigmentation prevention, reduction, improvement or treatment agent using the skin model of the present invention. More specifically, the screening method comprises the steps of: Culturing the skin model according to the present invention in a medium containing a candidate component; Identifying the location of melanin accumulation in keratinocytes; A step of comparing the melanin accumulation location in keratinocytes of a skin model cultured in a control medium containing no candidate component, and selecting a candidate component that causes a change in the accumulation location as an agent for preventing, reducing, improving or treating pigmentation. Includes. In such a screening method, the effect of the candidate ingredient that brings about a change in the accumulation location can be determined depending on the skin model used. The accumulation location in cultured keratinocytes that differs only in that it does not contain the candidate ingredient can be used as the melanin accumulation location in keratinocytes. A control group may be previously subjected to an experiment and the accumulation location recorded.

[0032] In order to capture the change in the melanin accumulation position, it is preferable to appropriately adjust the gel hardness and the unevenness parameter of the extracellular matrix protein gel in the skin model used in such a screening method. The skin model using the extracellular matrix protein gel with deep unevenness corresponds to the skin of the blemished area. The skin of the blemished area has a basement membrane with deep unevenness, and in such a skin model, the melanosomes taken up by the keratinocytes are accumulated around the nucleus. Therefore, it is preferable to use a skin model prepared using an extracellular matrix protein gel prepared with an unevenness parameter of #180 to #280. Furthermore, since the melanin synthesis is high and the difference in distribution is easily visible when the gel hardness is softened, it is preferable to use a skin model prepared using an extracellular matrix protein gel with a gel hardness of 10 to 50 kPa. By using such a skin model and culturing it with a medium containing a candidate component, when the accumulation position of the melanosomes changes from the perinucleus to the periphery of the membrane, the candidate component can be screened as a preventive, mitigating, improving or therapeutic agent for pigmentation.

[0033] Furthermore, in another aspect, the present invention may relate to a method for screening for a pigmentation prevention, reduction, improvement or treatment agent based on a melanin synthesis index. Such a screening method includes the steps of: Cultivating a skin model comprising melanocytes and keratinocytes in a medium containing a test drug; Identifying indicators of melanin synthesis in melanocytes; and A step of comparing the melanin synthesis index in melanocytes of the skin model cultured in a control medium containing no candidate component, and selecting a candidate component whose melanin synthesis index shows a decrease in melanin synthesis as a pigmentation reducing agent or pigmentation pigmentation preventing agent. Examples of melanin synthesis indicators include melanin itself, expression of melanin synthesis-related genes, melanin transport-related genes, protein expression, and the number and length of melanocyte dendrites. Any gene known in the art may be used as the melanin synthesis-related gene, and examples thereof include MLANA, TRP1, gp100, DCT, and tyrosinase. Any gene known in the art may be used as the melanin transport-related gene, and examples thereof include MREG and MLPH. The expression of melanin synthesis-related genes in melanocytes may be detected by isolating melanocytes, or may be detected together with keratinocytes. In another embodiment, the expression may be detected using a fluorescent microscope or the like in a skin model.

[0034] The pigmentation preventing, reducing, improving or treating agent screened in the present invention can be referred to as an agent for preventing, reducing, improving or treating spots, an agent for preventing, reducing, improving or treating dullness, an agent for preventing, reducing, improving or treating freckles, or an agent for preventing, reducing, improving or treating uneven skin tone.

[0035] In order to capture the expression change of melanin synthesis-related genes, it is preferable to appropriately adjust the gel hardness and unevenness parameters of the skin model used in the screening method. Since the amount of melanin production is high at a certain relatively low gel hardness, it is preferable to use a skin model prepared using an extracellular matrix protein gel with a gel hardness of 10 to 50 kPa. Furthermore, a skin model using an extracellular matrix protein gel with deep unevenness corresponds to skin at a spot. Therefore, from the viewpoint of screening for a spot prevention, reduction, improvement or treatment agent, it is preferable to use a skin model prepared using an extracellular matrix protein gel prepared with an unevenness parameter of #180 to #280. By using such a skin model and culturing it with a medium containing a candidate component, if the expression of a melanin synthesis indicator is reduced, the candidate component can be screened as a spot prevention, reduction, improvement or treatment agent.

[0036] Candidate components used in the screening method of the present invention can be any library of cosmetic materials, food materials, pharmaceutical materials, etc. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or may be synthesized compounds and prepared extracts.

[0037] The pigmentation prevention, reduction, improvement or treatment agent screened by the present invention may be incorporated into cosmetics, pharmaceuticals or quasi-drugs, and may also be incorporated into foods, such as nutritional supplements such as supplements. These drugs may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated into skin topical preparations. The skin topical preparation is not particularly limited as long as it can be applied to the skin, and any dosage form can be applied, such as a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into a skin topical preparation, a base and excipients, such as a preservative, emulsifier, pH adjuster, etc., that are commonly used in skin topical preparations may be used. When blended in cosmetics, it can be blended in face or body cosmetics such as lotions, milky lotions, beauty essences, creams, lotions, packs, essences, gels, etc., makeup cosmetics such as foundations, makeup bases, concealers, etc., and even bath additives, etc. By using cosmetics, medicines, and quasi-drugs containing the pigmentation preventing, reducing, improving, or treating agent screened by the present invention, it is possible to suppress melanin production or exert a whitening effect, and to prevent, reduce, improve, or treat pigmentation.

[0038] The pigmentation reducing or improving agent screened by the present invention can be arbitrarily selected in concentration from the viewpoint of exerting the desired effect, for example, whitening effect and / or blemishes, dullness, freckles, and / or uneven skin tone improving effect. From the viewpoint of formulation as a skin external preparation, the present invention can be formulated at 0.0005% to 0.5%. From the viewpoint of fully exerting the effect, it can be formulated preferably at 0.001% or more, and more preferably at 0.005% or more. From the viewpoint of avoiding a strong odor, it can be formulated preferably at 0.1% or less, and more preferably at 0.05% or less.

[0039] All documents mentioned herein are incorporated by reference in their entirety.

[0040] The following examples of the present invention are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. The present invention can be modified, for example, by adding, deleting, and replacing the constituent elements of the present invention, without departing from the spirit of the present invention. EXAMPLES

[0041] Example 1: Preparation of extracellular matrix protein gel Using quantum beam nanoimprinting technology, gelatin was crosslinked with gamma rays without the use of drugs, gelled, and at the same time, an extracellular matrix protein gel with a specified unevenness parameter was created using a mold. The molds used were PDMS molds transferred from the surfaces of sandpaper with grits of 600 (#600) and 280 (#280). Polydimethylsiloxane (PDMS) containing a base material and a hardener was poured onto the surface of the sandpaper, heated to harden it, and then peeled off to create a PDMS mold with an irregular unevenness structure similar to that of sandpaper. Next, a gelatin solution with an initial concentration of 10% was poured into a cell culture dish, a PDMS mold with a specified unevenness parameter was placed on it, and gamma rays were irradiated at a dose rate of 5 kGy / h. The resulting gel was immersed in PBS and incubated at 37°C, and the compressive elastic modulus of the gel was measured using a rheometer device, confirming that it reached 20 kPa and 80 kPa.

[0042] Example 2: Melanosome uptake in a skin model Keratinocytes obtained from skin of a 0-year-old child were seeded on the extracellular matrix protein gels with a compressive modulus of 20 kPa and transferred from the mold #600 prepared in Example 1, and on the extracellular matrix protein gels with a compressive modulus of 20 kPa and transferred from the mold #280, and monolayer cultured overnight in Epilife (Kurabo) medium. The next day, melanosomes isolated from mouse melanoma cells B16 were added to the medium at a concentration of 1:40 and cultured for one day to be incorporated into the keratinocytes. The next day, the gels were fixed with PFA fixative. After washing with PBS, nuclear staining was performed with DAPI and observed under a confocal microscope (LSM880, Zeiss) (Figures 1(A)(B)).

[0043] The number of cells containing melanosomes was counted by dividing them into those localized around the cell membrane and those around the nucleus, and quantification was performed using Fiji (number of samples = 11-12, total number of cells in each sample = 50-250) (Figure 1C). Results: When using the extracellular matrix protein gel (fine unevenness) transferred from mold #600, melanosomes were localized around the cell membrane (Figure 1A). On the other hand, when using the extracellular matrix protein gel (deep unevenness) transferred from mold #280, melanosomes were found more frequently around the nucleus (Figure 1B). Furthermore, quantification confirmed a significant difference in localization (significant differences exist when the alphabets are different, P<0.0001) (Figure 1C).

[0044] Example 3: Melanin production in a skin model Keratinocytes (cell number: 1×10 ) obtained from 0-year-old skin were placed on the extracellular matrix protein gels with a compressive modulus of 20 kPa and transferred from the mold #280 prepared in Example 1, and on the extracellular matrix protein gels with a compressive modulus of 80 kPa and transferred from the mold #280. 5 cells / well) and melanocytes isolated from the skin of a 22-year-old (cell number: 2 × 10 4cells / well) were co-cultured in CnT-PR-KM (CellnTec) medium for 3 days. The gels were then fixed with PFA fixative. After washing with PBS, immunostaining was performed using MLANA antibody (ab234416) and TRP1 antibody (ab3312). Then, the cells were fluorescently labeled with AlexaFluor647 and Alexa Fluor488, and nuclear stained with DAPI, and observed under bright field conditions with a confocal microscope (LSM880, Zeiss) (A) and (B). Fluorescence at 405 nm (DAPI), 488 nm (TRP1), and 647 nm (MLANA) was then observed. The fluorescence intensity of MLANA and TRP1 in the cells (number of samples = 4, number of cells in each sample = 10) was quantified using Fiji. Bright field observation showed that the degree of blackening was higher at 20 kPa (soft gel) than at 80 kPa (hard gel) (Figures 2(A) and (B)). The fluorescence intensity of MLANA and TRP1 was significantly higher at 20 kPa (soft gel) than at 80 kPa (hard gel) (p<0.005).

Claims

1. A skin model with controlled pigmentation, comprising an extracellular matrix protein gel having an uneven surface, keratinocytes seeded on the gel, and further melanosomes or seeded melanocytes.

2. The skin model according to claim 1, wherein pigmentation is regulated by the localization of melanosomes taken up by keratinocytes around the nucleus and / or around the cytoplasm and cell membrane.

3. The skin model according to claim 1, wherein the extracellular matrix protein gel having the uneven surface is a gel transferred from a mold having predetermined unevenness parameters.

4. The skin model according to claim 3, wherein the extracellular matrix protein gel having an uneven surface is a gel produced by curing a protein aqueous solution or physical gel by irradiating it with a quantum beam while a mold having predetermined unevenness parameters is in contact with it, and then peeling it off the mold.

5. The skin model according to claim 1, wherein the pigmentation-controlled skin model is configured such that the surface roughness parameter of the extracellular matrix protein gel and the hardness parameter of the extracellular matrix protein gel are adjusted to match a predetermined melanin distribution and / or melanin synthesis in a cell culture obtained by culturing the cells with the gel.

6. The skin model according to claim 5, wherein the protein is prepared such that the roughness parameters of the extracellular matrix protein gel are a roughness depth of 0 μm or more and 200 μm or less, and a roughness width of 0 μm or more and 1500 μm or less.

7. The skin model according to claim 5, wherein the unevenness parameter of the extracellular matrix protein gel is achieved on an uneven surface obtained by transferring the extracellular matrix protein gel to a mold having a surface roughness of sandpaper grit (particle size) of #12 or higher and #2000 or lower (in accordance with JIS R 6010).

8. A skin model according to claim 1 for comparative studies of pigmented and non-pigmented areas.

9. The skin model according to claim 1, wherein the protein is prepared such that the hardness parameter of the extracellular matrix protein gel is hard between 0.5 kPa and 200 kPa when measured by any of the methods selected from static testing, transverse vibration testing, ultrasonic testing, rheometer testing, nano-microindentation testing, and coherence elastography (OCE).

10. A method for manufacturing a skin model, A process of obtaining a laminate by stacking molds having a surface roughness of sandpaper grit #12 to #2000 (according to JIS R 6010) onto an aqueous solution or physical gel for preparing an extracellular matrix protein gel; A step of irradiating the laminate with a quantum beam of 5 to 50 kGy; A step of removing the mold from the irradiated laminate to obtain an extracellular matrix protein gel having an uneven surface; The extracellular matrix protein gel is immersed in phosphate-buffered saline (PBS) or culture medium, incubated at 37°C, and uncrosslinked components are removed; A step of seeding keratinocytes onto the extracellular matrix protein gel, and adding melanosomes or melanin, or seeding melanocytes; The process of culturing cells in a culture medium; and The process of culturing cells by exposing them to the gas phase. The manufacturing method, including the above.

11. A skin model prepared by the manufacturing method described in claim 10.

12. A screening method for agents that prevent, reduce, improve, or treat hyperpigmentation, A step of culturing the skin model according to claims 1 to 9 and 11 in a culture medium containing candidate components; A step to identify the location of melanin accumulation in keratinocytes; A process of comparing the location of melanin accumulation in keratinocytes of a skin model cultured in a control medium that does not contain the candidate component, and selecting candidate components that cause changes in the accumulation location as agents for preventing, reducing, improving, or treating hyperpigmentation. The screening method, including the above.

13. A screening method for agents that prevent, reduce, improve, or treat hyperpigmentation, A step of culturing melanocytes seeded in the skin model according to claims 1 to 9 and 11 in a culture medium containing a test drug; A process for identifying indicators of melanin synthesis in melanocytes; A process of selecting candidate components for use in preventing, reducing, improving, or treating hyperpigmentation by comparing the melanin synthesis index in melanocytes of a skin model cultured in a control medium that does not contain the candidate component with that of a candidate component. The screening method, including the above.

14. The screening method according to claim 13, wherein the melanin synthesis index is the amount of melanin.

15. The screening method according to claim 13, wherein the melanin synthesis index is the amount of protein or expression level of a melanin synthesis-related gene.

16. The screening method according to claim 15, wherein the melanin synthesis-related gene is selected from the group consisting of MLANA, PMEL, TYR, DCT, and TRP1.

17. The screening method according to claim 13, wherein the activation state of melanocytes as melanin-producing cells is indicated by an increase in the number and length of dendrites in correlation with the aforementioned melanin synthesis index.

18. The screening method according to claim 13, wherein the activation state of melanocytes as melanin transport ability is used as an indicator by increasing the transfer of melanin from melanocytes to keratinocytes in correlation with the melanin synthesis index, and the indicator of melanin transport ability is the amount of protein or expression level of a group of melanin transport-related genes consisting of MREG and MLPH.

19. A method for producing an extracellular matrix protein gel for keratinocyte culture, wherein an aqueous solution of extracellular matrix protein or a physical gel is used. A process of obtaining a laminate by laminating molds having a surface roughness of #12 or higher and #2000 or lower (in accordance with JIS R 6010) using sandpaper; A step of irradiating the extracellular matrix protein gel of the laminate with a quantum beam of 5 kGy or more and 50 kGy or less; A step of removing the mold from the irradiated laminate to obtain an extracellular matrix protein gel having an uneven surface; The manufacturing method, including the above.

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