Artificial skin model and its manufacturing method

Abstract

To develop a technology for easily and quickly mass-producing artificial skin models of any shape that reproduce the reality of real skin. [Solution] An artificial skin model having an arbitrary shape, including a culture scaffold made of alginate hydrogel molded into an arbitrary shape and cultured skin tissue on the culture scaffold, and a method for manufacturing an artificial skin model, including the steps of: using a 3D modeling machine to create a mold with the shape of the target skin carved out; gelling sodium alginate in the created mold to create an alginate hydrogel; and removing the created alginate hydrogel from the mold, culturing skin cells using the alginate hydrogel as a culture scaffold, inducing differentiation, and reconstructing skin tissue.

Description

[Technical Field]

[0001] The present invention relates to an artificial skin model having any shape, a method for producing the same, and a method for using the same. [Background technology]

[0002] Recent advances in tissue engineering technology have led to the development of artificial tissue models that mimic in vivo tissues. Artificially constructed tissue models are useful not only for organ replacement in regenerative medicine but also for evaluating the efficacy and safety of drugs, cosmetics, and health foods. Therefore, there is great potential for the development of tissue construction technologies that more closely replicate the structure and function of living tissues. In particular, autologous cultured epidermis, which is created by culturing epidermal stem cells in vitro and reconstructing tissue with a structure similar to that of living epidermal tissue, has been rapidly adopted as a regenerative medicine product. In addition to cultured epidermis, research has also been conducted on more advanced artificial skin models, such as cultured skin models with a dermal layer. These models are used in medical settings for wound closure in burns and skin ulcers. They are also used in cosmetic and pharmaceutical development as tools to predict the skin irritation and toxicity of test substances and evaluate their efficacy without the use of laboratory animals.

[0003] Today, many companies both in Japan and overseas are selling commercially available artificial skin models with cultured epidermis and dermis layers, making it possible to conduct safety evaluation tests, such as skin irritation tests, in vitro. In recent years, a method for producing cultured epidermal models using immortalized keratinocytes has been reported to enable stable evaluation without lot-to-lot variation (Patent Document 1). Examples of the use of these artificial skin models are becoming more widespread. For example, one study reproduced a rough skin condition by exposing the stratum corneum surface of the model to SDS (sodium dodecyl sulfate) (Patent Document 2), and another reported development of a model in which the degree of roughness was controlled by combining genome editing technology (Patent Document 3). However, conventional artificial skin models are primarily reconstructed within culture inserts, resulting in a flat, sheet-like structure that does not adequately reproduce the realism of the appearance and function of actual living skin.

[0004] In recent years, artificial skin has been developed as a covering material for humanoid robots, such as covering the periphery of finger-type robots (Non-Patent Document 1) and glove-shaped artificial skin (Non-Patent Document 2). However, their fabrication requires specialized equipment and a continuous culture medium supply system to mold them into the desired shape, resulting in complex processes and long cultivation periods. Furthermore, the development of artificial skin for cosmetics and humanoid robots requires high levels of realism in terms of appearance, texture, and feel, but no technology for fabricating such artificial skin has yet been established. Therefore, there is a need for a technology that can easily and quickly mass-produce artificial skin models of any shape that more closely resemble the realism of real skin.

[0005] On the other hand, hydrogels are swollen bodies in which polymers forming a three-dimensional network structure retain a large amount of water, and their use as biomaterials is increasing. Among these, alginate gels, whose main chain is alginic acid extracted from seaweed such as kelp and wakame, have attracted attention due to their high biocompatibility. To date, a method for preserving cells and tissues in a liquid composition containing alginate gel and deacylated gellan gum has been reported (Patent Document 4), and a medical material in which chondrocytes and other cells are encapsulated in a fibrous aggregate made of alginate gel fiber has been reported (Patent Document 5). However, there have been no examples of applying alginate hydrogels as cell culture scaffolds or artificial skin models. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2018-102186 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-237098 [Patent Document 3] Japanese Patent Publication No. 2020-92628 [Patent Document 4] WO2019 / 049985 publication [Patent Document 5] Japanese Patent Application Publication No. 2023-157891 [Non-patent literature]

[0007] [Non-Patent Document 1] Matter. 2022 July;5, 2190-2208, Living skin on a robot [Non-patent document 2] Sci Adv. 2023 Jan 27;9(4):eade2514, Engineering edgeless human skin with enhanced biomechanical properties Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, in view of the above-mentioned circumstances, the object of the present invention is to develop a technology that can easily and quickly provide a large number of artificial skin models that have any shape and reproduce the reality of real skin. [Means for solving the problem]

[0009] The inventors conducted extensive research to solve the above-mentioned problems and have succeeded in producing an artificial skin model in a short period of time by using a 3D modeling machine to create a mold with the shape of the target skin carved out, controlling the solidification time to produce alginate hydrogel within the mold, and using this as a culture scaffold to induce differentiation of epidermal stem cells and dermal stem cells. This precisely reproduces the shape of the target skin and provides a highly realistic appearance, texture, and feel. Furthermore, it has been confirmed that the artificial skin model of the present invention is capable of visualizing areas of skin damage caused by the direction and intensity of ultraviolet light irradiation, and capturing changes in facial shape due to the direction of gravity. The present invention was completed based on these findings.

[0010] That is, the present invention includes the following inventions. (1) An artificial skin model having an arbitrary shape, comprising a culture scaffold made of alginate hydrogel molded into an arbitrary shape and cultured skin tissue on the culture scaffold. (2) The artificial skin model described in (1), wherein the alginate hydrogel is a gel formed from a poorly soluble calcium salt of sodium alginate. (3) The artificial skin model described in (2), wherein the poorly soluble calcium salt is calcium sulfate. (4) The artificial skin model described in (1), wherein the alginate hydrogel is a gel formed from a poorly soluble calcium salt of sodium alginate and a chelating agent. (5) The artificial skin model according to (4), wherein the chelating agent is sodium pyrophosphate. (6) The artificial skin model described in (1), wherein the alginate hydrogel is a gel of sodium alginate with calcium sulfate and sodium pyrophosphate. (7) The artificial skin model described in (1), wherein the cultured skin tissue includes epidermal tissue and dermal tissue. (8) The artificial skin model described in (7), wherein the cultured skin tissue is reconstructed from epidermal keratinocytes and dermal fibroblasts. (9) The artificial skin model described in (8), wherein the epidermal keratinocytes and fibroblasts are reconstructed from epidermal stem cells or progenitor cells and dermal stem cells or progenitor cells. (10) The artificial skin model according to (9), wherein the stem cells or progenitor cells are immortalized stem cells or progenitor cells. (11) The artificial skin model described in (9), wherein the stem cells are stem cells derived from hair follicles. (12) An artificial skin model that includes a culture scaffold made of alginate hydrogel molded into any shape and cultured skin tissue on top of the culture scaffold, reproducing the color of living skin. (13) The artificial skin model according to (12), wherein the alginate hydrogel contains one or two components selected from hemoglobin and beta-carotene, which determine skin color. (14) The artificial skin model described in (12), wherein the cultured skin tissue has epidermal tissue containing melanocytes. (15) The artificial skin model described in (12), characterized in that the alginate hydrogel contains one or two components selected from hemoglobin and beta-carotene, which determine skin color, and the cultured skin tissue has epidermal tissue containing melanocytes. (16) The artificial skin model according to (1), wherein the shape of the artificial skin model is the shape of a face. (17) The artificial skin model described in (1), wherein the artificial skin model is used for evaluation tests of the efficacy and safety of ingredients of pharmaceuticals, quasi-drugs, or cosmetics. (18) The artificial skin model described in (1), wherein the artificial skin model is for skin transplantation. (19) The artificial skin model according to (1), wherein the artificial skin model is for covering a humanoid robot. (20) A method for producing an artificial skin model, comprising the following steps: (a) A process of creating a mold with the shape of the target skin carved out using a 3D modeling machine. (b) A step of gelling sodium alginate in the mold prepared in step (a) to prepare an alginate hydrogel. (c) A step of removing the alginate hydrogel prepared in step (b) from the mold, culturing skin cells using the alginate hydrogel as a culture scaffold, and inducing differentiation to reconstruct skin tissue. (21) A method for visualizing areas of cell damage caused by ultraviolet light by irradiating an artificial skin model having the facial shape described in (16) with ultraviolet light and staining the cells in the skin model after irradiation. (22) A method for analyzing changes in skin shape due to changes in the direction of gravity using an artificial skin model having the facial shape described in (16). (23) A humanoid robot that is covered with the artificial skin model described in (1) and has the texture and elasticity of living skin. [Effects of the Invention]

[0011] The alginate hydrogel used in the artificial skin model of the present invention combines high moldability, water absorption, flexibility similar to that of real living skin, and heat resistance that allows for autoclave sterilization to prevent bacterial contamination, making it ideal as a culture scaffold. Furthermore, since alginate is extracted from natural seaweed, it is highly safe and has a low environmental impact. While conventional artificial skin models require approximately one month to produce, the present invention enables the production of artificial skin with sufficient tissue development and skin barrier function in approximately two weeks. Furthermore, the method for producing the artificial skin model of the present invention requires fewer substrates and processes. Simply creating a single mold of any desired shape using a 3D modeling machine allows for the simple and rapid mass production of artificial skin models using alginate hydrogel as a culture scaffold.

[0012] Furthermore, the artificial skin model of the present invention can be used to create an artificial skin model that reproduces cell damage to skin caused by ultraviolet rays and changes in skin shape due to changes in the direction of gravity. Therefore, by using a model that reproduces such skin damage and environmental changes, it becomes possible to screen and evaluate the effectiveness of UV protection agents, whitening ingredients, and ingredients that improve wrinkles and sagging. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows an image of the culture of epidermal stem cells and dermal stem cells used to prepare the artificial skin model of the present invention. [Figure 2] Figure 2 shows a schematic diagram of the collagen gel used to form cultured skin tissue in the artificial skin model of the present invention, as well as images of confluent epidermal stem cells on the surface of the collagen gel and dermal stem cells cultured inside the collagen gel. [Figure 3] Figure 3 shows the three-dimensional information of the facial shape of the biological model (donor) imported into the 3D modeling software. [Figure 4] Figure 4 shows the appearance of a culture scaffold (made of urethane) created using a 3D modeling machine. [Figure 5] FIG. 5 shows a mold (blueprint) with the shape of the face of a living model (donor) carved out. [Figure 6] Figure 6 shows the appearance of the cast alginate hydrogel. [Figure 7] FIG. 7 shows the flow of producing the artificial skin model of the present invention. [Figure 8] FIG. 8 shows the appearance of the artificial skin model of the present invention. [Figure 9] FIG. 9 shows the results of staining a thin section of the artificial skin model of the present invention. [Figure 10] FIG. 10 shows images of hair follicle epithelial stem cells that migrated from the plucked hair follicle and the cultured cells after subculture. [Figure 11] Figure 11 shows the appearance of an artificial skin model created using hair follicle epithelial stem cells. [Figure 12]FIG. 12 shows a schematic diagram and photographs of the UVB irradiation conditions (conditions when placed horizontally on the back and conditions when placed vertically) for the artificial skin model (face shape). [Figure 13] FIG. 13 shows the results of an MTT staining test on an artificial skin model that was irradiated with UVB while placed horizontally on its back or while standing vertically, as well as an artificial skin model that was not irradiated with UVB. [Figure 14] FIG. 14 shows a schematic diagram and photographs of UVB irradiation conditions (summer conditions, winter conditions) for an artificial skin model (face shape). [Figure 15] FIG. 15 shows the results of an MTT staining test on an artificial skin model irradiated with UVB under summer or winter conditions, and an artificial skin model not irradiated with UVB. [Figure 16] FIG. 16 shows a schematic diagram and photographs of the gravity direction conditions (when placed horizontally on the back and when standing vertically) for the artificial skin model (face shape). [Figure 17] Figure 17 shows images of the artificial skin model taken with a three-dimensional shape analysis camera when placed horizontally on its back and when standing vertically (circle: face line). [Figure 18] Figure 18 shows the appearance of a skin-colored artificial skin model created by culturing epidermal stem cells together with melanocytes and inducing their differentiation on a culture scaffold made of alginate hydrogel containing components that determine skin color (hemoglobin, beta-carotene). DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. Artificial Skin Model The artificial skin model of the present invention comprises a culture scaffold made of alginate hydrogel molded into any shape, and cultured skin tissue on top of the culture scaffold.

[0015] (Alginate hydrogel) Alginate hydrogels are hydrogels obtained by forming salts between alginate and divalent metal ions. The alginate hydrogels of the present invention are preferably hydrogels in which the gelation time of alginate is controlled by controlling calcium ionization (hereinafter, sometimes referred to as "ionization-controlled alginate hydrogels" in this specification). Specifically, the alginate hydrogels used in the present invention are preferably gels of sodium alginate with a poorly soluble calcium salt. Examples of poorly soluble calcium salts include calcium sulfate, calcium citrate, calcium carbonate, monocalcium phosphate, and dicalcium phosphate, with calcium sulfate being preferred. Here, the term "hydrogel" refers to a polymeric substance that forms a three-dimensional network structure by crosslinking polymer chains of water-soluble polysaccharides or proteins to make it insoluble in water, and that encapsulates a large amount of water within the network structure. The alginate hydrogel used in the artificial skin model of the present invention functions as a scaffold for culturing skin cells to reconstruct skin tissue and is excellent in moldability, medium water absorption, heat resistance, flexibility, and economy. On the other hand, the hydrogel used for the culture scaffold is not limited to alginic acid, and other water-soluble polysaccharides that gel with metal salts in the same way as alginic acid can also be used.

[0016] In addition to the poorly soluble calcium salt, it is preferable to use a chelating agent to control calcium ionization. Phosphate or EDTA can be used as the chelating agent, with phosphate being preferred and sodium pyrophosphate being more preferred.

[0017] (Cultured skin tissue) The cultured skin tissue in the artificial skin model of the present invention has a two-layer structure in which epidermal tissue is formed on top of dermal tissue, and is reconstituted by culturing epidermal stem cells or progenitor cells and dermal stem cells or progenitor cells.

[0018] The shape of the artificial skin model of the present invention is not particularly limited as long as it mimics the shape of the skin of a living body, and examples include the shape of a face, fingers, upper and lower limbs, and the shape of the entire head.

[0019] 2. Manufacturing method of the artificial skin model The method for producing an artificial skin model of the present invention includes the steps of: (a) creating a mold with the shape of the target skin carved out using a 3D modeling machine; (b) gelling sodium alginate in the mold created in step (a) to create an alginate hydrogel; and (c) removing the alginate hydrogel created in step (b) from the mold, culturing skin cells using the alginate hydrogel as a culture scaffold, inducing their differentiation, and reconstructing skin tissue.

[0020] Process (a): In step (a), the target skin shape, e.g., a human face shape, is scanned with a 3D scanner to obtain 3D data. This data is then used to create a mold with a cutout of the human face shape using a 3D modeling machine. The 3D modeling machine is a cutting device capable of applying shapes and patterns to resin-based materials. Commercially available 3D modeling machines (such as those manufactured by Roland DG Corporation) can be used. The mold material is not particularly limited as long as it has a certain degree of hardness, but examples include polyamide resin, polyethylene terephthalate (PET) resin, acrylonitrile-styrene copolymer (AS resin), acrylonitrile-butadiene-styrene copolymer (ABS resin), polyurethane resin, and polycarbonate resin. Alternatively, molds can be created using a 3D printer, which extrudes molten resin from a nozzle and builds it up to form a model, rather than cutting. Furthermore, by using a 3D bioprinter, which sprays cells and biomaterials from a nozzle like ink and layers them on top of each other, it is possible to directly mold a culture scaffold of any shape without using a mold.

[0021] Process (b): In step (b), sodium alginate is gelled in the mold prepared in step (a) to produce an alginate hydrogel. Mixing a sodium alginate solution with a calcium solution results in instantaneous gelation at room temperature, making it difficult to mold into any desired shape. Therefore, gelation in this step is carried out using a method in which calcium with controlled ionization is applied to sodium alginate to control the gelation time (an ionization control method). Specifically, a poorly soluble calcium salt is used to gel sodium alginate. A poorly soluble calcium salt is a calcium salt with a solubility in water at 25°C of 0.05 g / 100 mL or more but less than 1.0 g / 100 mL. Examples of poorly soluble calcium salts include calcium sulfate, calcium citrate, calcium carbonate, monocalcium phosphate, and dicalcium phosphate, with calcium sulfate being preferred. These poorly soluble calcium salts may be used alone or in combination.

[0022] The concentration of the sodium alginate solution for preparing alginate hydrogel is preferably 2.0% to 7.0% (w / v), more preferably 3.0% to 5.0% (w / v), in terms of the viscosity of the sodium alginate solution, the moldability and flexibility of the culture scaffold to be prepared, and the miscibility with poorly soluble calcium salts and chelating agents.

[0023] The concentration of the poorly soluble calcium salt solution to be mixed with the sodium alginate solution and used to gel the sodium alginate is preferably 1.5% to 7.5% (w / v), more preferably 3.0% to 5.0% (w / v).

[0024] Furthermore, in order to control the gelation time, it is more preferable to use a chelating agent when mixing the poorly soluble calcium salt solution with the sodium alginate solution. Phosphate or EDTA can be used as the chelating agent, with phosphate being preferred, and sodium pyrophosphate being more preferred. The concentration of the chelating agent solution is preferably 0.02% to 0.40% (w / v). By using the ionization control method, the sodium alginate solution can be allowed to stand at room temperature for about four hours, gradually gelling (solidifying) from a liquid state over time.

[0025] Process (c): In step (c), the alginate hydrogel prepared in step (b) is removed from the mold, and skin cells are cultured on the alginate hydrogel as a culture scaffold to induce differentiation and reconstruct skin tissue. The reconstruction of skin tissue can be carried out essentially according to a conventional method for producing three-dimensional cultured skin commonly used in the field, except that a culture vessel such as a culture insert with a liquid-permeable membrane on the bottom is used as the culture scaffold, and the alginate hydrogel prepared in step (b) is used. Here, the reconstruction of skin tissue refers to the formation of an epidermal layer in which epidermal keratinocytes are stratified by exposure to air on a dermal layer containing fibroblasts.

[0026] Commercially available epidermal keratinocytes and fibroblasts can be used as skin cells to be cultured on an alginate hydrogel scaffold. Primary cultured epidermal keratinocytes and fibroblasts are particularly preferred. Stem cells and progenitor cells isolated from these cells using FACS or other methods based on the expression of markers to assess the undifferentiated state of stem cells can also be used. The origin of the skin cells is not particularly limited as long as they are mammalian, and examples include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses, with humans being preferred. Furthermore, immortalized skin cells that proliferate indefinitely while maintaining stem cell properties may also be used. The immortalization method is not limited as long as it immortalizes cultured cells without inducing cell death. Examples include introducing an immortalization gene into primary cultured epidermal keratinocytes and fibroblasts. Here, the term "immortalizing gene" refers to a gene that immortalizes cells and gives them the ability to proliferate indefinitely, and examples thereof include the telomerase reverse transcriptase (TERT) gene, genes that regulate the expression or activity of telomerase (e.g., Myc gene, Ras gene, etc.), and viral genes (SV40T, HPV E6-E7, EBV, etc.), with the telomerase reverse transcriptase (TERT) gene being preferred, and the human telomerase reverse transcriptase (hTERT) gene being more preferred.

[0027] The production of three-dimensional cultured skin consists of a proliferation and culture step and a differentiation induction step. In a preferred embodiment of the present invention, the proliferation and culture step is carried out using a collagen gel, a collagen sponge, or acellular dermis from which the epithelium and fibroblasts have been removed as a support, and fibroblasts may also be incorporated into these supports as appropriate.

[0028] In one embodiment, dermal stem cells are added to a collagen solution and cultured to form a dermal layer in which the dermal stem cells are embedded in the collagen gel, and then epidermal stem cells are seeded thereon and further cultured to proliferate the epidermal stem cells until they become confluent, thereby forming an epidermal layer. The number of epidermal stem cells to be added is not particularly limited, but is preferably 15 x 10 4 ~120×10 4 cells / cm2 is preferred, and 30 × 10 4 ~90×10 4 cells / cm 2 is more preferable. The proliferation culture is carried out for, for example, 1 to 6 days, preferably 2 to 4 days. During this period, the medium may be replaced as appropriate. Whether the proliferated epidermal stem cells are in a confluent state can be confirmed using a cell staining reagent such as the CnT-ST-100 stain kit (manufactured by CELLnTEC).

[0029] The cell growth medium is not particularly limited as long as it is a basal medium suitable for the growth and subculture of epidermal keratinocytes, but a serum-free, low-calcium basal medium is preferred, and commercially available media such as MCDB153 medium (Sigma), HuMedia-KG2 (Kurabo), serum-free medium for normal human epidermal keratinocytes (DS Pharma Biomedical), and Keratinocyte-SFM (Thermo Fisher Scientific) may be used. The medium may also contain growth factors such as basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), and stem cell factor (SCF). To increase the proliferation rate, the medium may contain epidermal growth factor (EGF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), L-glutamine, fibronectin, progesterone, selenite, B27 supplement, N2 supplement, or ITS supplement, as needed. Antibiotics may also be added as needed. The calcium concentration of the cell proliferation medium is preferably about 0.03 to 0.15 mM.

[0030] Next, in the differentiation induction process, the collagen gel containing the epidermal and dermal layers is mounted on an alginate hydrogel, and the medium is changed to a cell differentiation medium and differentiation induction culture is carried out for approximately 8 to 10 days. For the final 1 to 2 days, the entire model is exposed to air (atmospheric air) to induce the final differentiation of stratified epidermal keratinocytes.

[0031] The cell differentiation medium is not particularly limited as long as it is a basal medium suitable for inducing differentiation into epidermal keratinocytes, and commercially available media such as CnT-Prime 3D Barrier Culture Medium (manufactured by CELLnTEC) may be used. The calcium concentration of the cell differentiation medium is preferably about 1.2 to 3.0 mM.

[0032] The culture temperature for proliferation and differentiation induction varies depending on the origin of the cells, but for example, in the case of cells of human origin, it is preferably 30 to 40° C., more preferably 36 to 38° C. Furthermore, the CO gas concentration is preferably, for example, about 1 to 10%, more preferably about 2 to 5%.

[0033] The artificial skin model of the present invention, manufactured through these processes, differs from conventional three-dimensional cultured skin models, which have a flat sheet-like structure, in that it is formed on a hydrogel that mimics the three-dimensional shape of actual biological skin, making it highly realistic in appearance, texture, and feel.

[0034] In another embodiment, an artificial skin model that reproduces the color of living skin can be produced by adding a pigment component that determines skin color to the alginate hydrogel in the above-mentioned step (b) of preparing the alginate hydrogel that serves as the culture scaffold. Pigment components that determine the color of living skin include, but are not limited to, hemoglobin and β-carotene, and these two components may be used in combination. The concentration of hemoglobin added to the culture scaffold is not particularly limited, but the final concentration of hemoglobin relative to the culture scaffold is preferably 0.001% to 1% (w / v), more preferably 0.01 to 0.1% (w / v). The concentration of β-carotene added to the culture scaffold is also not particularly limited, but the final concentration of β-carotene relative to the culture scaffold is preferably 0.0005% to 0.5% (w / v), more preferably 0.005% to 0.05% (w / v).

[0035] In yet another embodiment, when seeding epidermal stem cells in the skin cell culture in step (c) above, melanocytes can also be seeded at the same time to produce an artificial skin model with epidermal tissue containing melanocytes. The melanocytes seeded may be of human or other animal origin, and may be primary cultures, established cell lines, or immortalized cell lines. The melanocytes seeded are preferably 0.1% to 10% of the epidermal stem cells seeded, and most preferably 1% to 5%.

[0036] 3. How to use the artificial skin model The artificial skin model of the present invention can be used as an alternative to animal testing to evaluate the efficacy and safety of cosmetics, topical skin preparations, and chemical substances (detergents, clothing dyes, etc.). For example, efficacy evaluations include skin barrier function, moisture or oil retention and regulation, prevention and improvement of wrinkles, sagging, and blemishes, and moisture permeability. Safety evaluations include the occurrence of erythema, redness, inflammation, pigmentation, swelling, and rashes. The artificial skin model of the present invention can also be used to screen substances that improve epidermal and dermal function. Improvements in epidermal function include improving the epidermal barrier function (e.g., moisture retention and prevention of external invasion of ultraviolet rays, chemicals, bacteria, etc.), normalizing skin turnover, and normalizing melanin metabolism. Improvements in dermal function include improvement of wrinkles and sagging.

[0037] As shown in the examples below, the artificial skin model of the present invention can be a model that accurately reproduces the degree of skin damage caused by different UV irradiation directions and different seasons. Therefore, the artificial skin model of the present invention can be used to evaluate the effects of UV rays by taking advantage of the three-dimensional shape of the face, develop whitening agents and sunscreens, predict the risk of UV-induced skin damage in each season, and provide seasonal beauty advice and counseling. As shown in the examples below, the artificial skin model of the present invention can also be a model that accurately reproduces changes in skin shape relative to the direction of gravity. Therefore, the artificial skin model of the present invention can be used in research on sagging and the development of methods for preventing aging. Furthermore, as shown in the examples below, the artificial skin model of the present invention has excellent not only appearance but also elasticity, and is expected to be useful as a covering material for humanoid robots, which require an appearance, texture, and feel that are extremely similar to human skin. Therefore, the artificial skin model of the present invention provides a new evaluation system and application fields that have not been seen before.

[0038] In the present invention, the safety and efficacy of cosmetics and pharmaceuticals can be evaluated and screened by contacting a test substance with the artificial skin model of the present invention and measuring changes in the epidermal cells of the model. In this case, a more accurate evaluation can be achieved by comparing the measurement results with a control artificial skin model of the present invention that has not been contacted with the test substance. Changes in epidermal cells include changes in their number, morphology, distribution, localization, migration, and loss, as well as changes in the expression levels of specific genes in epidermal cells (e.g., epidermal keratinocyte differentiation marker genes such as involucrin, filaggrin, and loricrin), and changes in the number of melanocytes. For example, if epidermal cell death or proliferation inhibition is used as an indicator, the test substance can be evaluated as a substance that irritates the epidermis. If promotion of epidermal cell proliferation or an increase in stratum corneum thickness is used as an indicator, the test substance can be screened as a candidate substance for skin turnover promoters. Furthermore, if reduction or loss of melanocytes is used as an indicator, the test substance can be screened as a candidate substance for skin whitening agents. For example, the test substance can be administered or applied from the top of the artificial skin model.

[0039] The measurement of changes in epidermal cells is not particularly limited and can be carried out by known methods, such as microscopic observation of changes in the number and morphology of epidermal cells, cell proliferation and viability tests using MTT, XTT, WST-8, Alamar Blue, etc., viable cell counting methods such as trypan blue dye exclusion tests, and cytotoxicity tests such as LDH assays.

[0040] Similarly, changes in dermal cells in the artificial skin model of the present invention can also be measured. Changes in dermal cells include changes in their number, morphology, distribution, localization, migration, disappearance, etc., as well as changes in the expression levels of genes involved in the construction of extracellular structures within dermal cells (e.g., collagen genes and elastin genes) and matrix metalloproteinases (MMPs), which are collagen-degrading enzymes. For example, if cell death or inhibition of proliferation of dermal cells is used as an indicator, the test substance can be evaluated as a substance that irritates the dermis. If promotion of dermal cell proliferation, an increase in collagen content, or a decrease in matrix metalloproteinases is used as an indicator, the test substance can be screened as a candidate substance for improving wrinkles and sagging.

[0041] Similarly, the measurement of changes in dermal cells is not particularly limited and can be carried out by known methods such as microscopic observation of changes in the number, morphology, etc. of dermal cells, cell proliferation / viability tests using MTT, XTT, WST-8, Alamar Blue, etc., viable cell counting methods such as trypan blue dye exclusion tests, and cytotoxicity tests such as LDH assays.

[0042] Test substances are primarily intended for use in cosmetics and / or pharmaceuticals. Examples include mixtures containing multiple compounds, such as extracts from animal or plant tissues or microbial cultures, and purified preparations thereof; naturally occurring molecules (e.g., amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, steroids, glycoproteins, proteoglycans, etc.); synthetic analogs or derivatives of naturally occurring molecules (e.g., peptidomimetics); and non-naturally occurring molecules (e.g., small organic compounds produced using combinatorial chemistry techniques); as well as mixtures thereof. Test substances may be tested individually, or mixtures (including libraries) of several candidate test substances may be tested. Libraries containing multiple test substances include synthetic compound libraries and peptide libraries. [Example]

[0043] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0044] (Example 1) Preparation of collagen gel used to prepare an artificial skin model Epidermal cells used in this experiment were commercially available normal human epidermal keratinocytes (Kurabo Industries, Ltd.) maintained in HuMedia-KG2 medium (Kurabo Industries, Ltd.). Dermal cells used in this experiment were commercially available normal human fibroblasts (Kurabo Industries, Ltd.) maintained in DMEM medium (Nacalai Tesque, Inc.) containing 10% FBS. These cells were cultured in 10 cm dishes, harvested with trypsin-EDTA, and immunostained with anti-CD271 antibody (Origene, Inc.), a stem cell marker. From the stained cells, the CD271-positive cell fraction was isolated using a FACS Melody (Becton Dickinson Japan), and epidermal stem cells and dermal stem cells were isolated. Figure 1 shows the culture images of epidermal stem cells and dermal stem cells isolated by FACS.

[0045] Next, using Cellmatrix Type I-A (Nitta Gelatin Co., Ltd.), a collagen gel preparation reagent, the dermal stem cells were separated by FACS to a concentration of 1 × 10 5 A collagen solution was prepared so that the density of cells / mL was reached. 10 mL of the collagen solution was poured into a 10 cm dish and incubated at 37°C for 30 minutes to produce a collagen gel with embedded dermal stem cells. Epidermal stem cells sorted by FACS were then applied to the surface of the collagen gel at a density of 1 × 10 6 Cells / mL were seeded and cultured in HuMedia-KG2 medium until the epidermal stem cells became confluent. Figure 2 shows images of epidermal stem cells that had reached confluence on the collagen gel surface and dermal stem cells inside the collagen gel.

[0046] We also established immortalized cell lines that maintain stemness and proliferate indefinitely by introducing immortalizing genes into commercially available normal human epidermal keratinocytes or normal human fibroblasts. To establish immortalized cell lines, we constructed vectors containing genes encoding three genes: the telomerase reverse transcriptase (TERT) gene (GenBank number: Nucleotide NM_198253.2), the cyclin-dependent kinase 4 (CDK4) gene (GenBank number: Nucleotide NM_000075.3), and the cyclin D1 (CCND1) gene. These vectors were then transfected into each cell, and single cells were isolated from the immortalized cell population by single-cell cloning. For the immortalized human epidermal keratinocytes obtained, the expression level of differentiation markers (FLG, IVL) when differentiated with 3 mM calcium chloride was used as an indicator, and for the immortalized human fibroblasts, the expression level of the collagen gene (COL1A1) when differentiated with TGFβ was used as an indicator to obtain single clonal lines of immortalized epidermal stem cell model cells and immortalized dermal stem cell model cells with high differentiation potential.

[0047] (Example 2) Selection of culture scaffold material We selected a culture scaffold material suitable for creating an artificial skin model that replicates the facial surface, a region with a complex three-dimensional structure. Urethane, styrene, and various hydrogels were examined as culture scaffold materials.

[0048] (1) Acquisition of facial shape data and 3D shape First, to create an artificial skin model with a precise facial shape, we used a 3D scanner VIVID910 (Konica Minolta) to obtain facial shape data of the subject. The obtained shape data was then imported into Rhinoceros 3D, a 3D modeling software. Figure 3 shows the 3D information of the facial shape imported into the software.

[0049] (2) Scaffold fabrication (2-1) Urethane or styrene Using a 3D modeling machine (Roland DG Corporation), we created scaffolds containing urethane or styrene. Figure 4 shows the urethane scaffolds we created. We were able to create scaffolds that precisely replicated the facial shape captured by a 3D scanner.

[0050] (2-2) Highly water-absorbent medium We investigated a method for creating a culture scaffold using a material with high medium water content. To achieve this, we utilized the facial shape data obtained in (1) to create a mold with a facial shape cut out of ABS resin material. Figure 5 shows the mold design. A 3D modeling machine was used to create a mold with a facial shape cut out of ABS resin, and we attempted to mold a culture scaffold by gelling (solidifying) the liquid material within this mold over time. The test materials used were commercially available collagen gel (Cellmatrix Type I-A: manufactured by Nitta Gelatin Co., Ltd.), gelatin (manufactured by BD), agar (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), agarose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), alginate gel, and alginate hydrogel.

[0051] The scaffold fabrication methods and performance results for the materials examined above are shown in Table 1 below.

[0052] [Table 1]

[0053] Because urethane and styrene have extremely low water absorption, attempts were made to reconstruct skin tissue using the collagen gel containing epidermal stem cells and dermal stem cells of Example 1, but it was difficult to achieve uniform reconstruction, demonstrating that they are insufficient as scaffold materials. Furthermore, similar results were obtained when reconstructing skin tissue using immortalized epidermal stem cells and dermal stem cells.

[0054] Collagen gel has low hardness and is not suitable for molding scaffolds of any shape. Furthermore, the gel dissolves during autoclaving to prevent bacterial contamination, making it unsuitable for culturing. Furthermore, highly pure collagen reagents are expensive, making them a cost disadvantage.

[0055] Although gelatin is inexpensive, it has low hardness and is not suitable for molding scaffolds of any shape, and the gel easily disintegrates under culture conditions.

[0056] Agar and agarose gel also lack sufficient heat resistance and moldability, making them unsuitable as scaffolds for artificial skin models.

[0057] Alginate gel was prepared by reacting a sodium alginate (Fujifilm Wako Pure Chemical Industries, Ltd.) solution with a calcium chloride solution. Although alginate gel has excellent heat resistance, the gelation reaction of alginate with calcium ions occurs instantaneously, making it difficult to mold the gel into any desired shape.

[0058] To control the gelation time of sodium alginate, we attempted to fabricate alginate hydrogels (ionization-controlled alginate hydrogels) using a technique called the ionization-control method, which involves adding a poorly soluble calcium salt and a chelating agent to a sodium alginate solution to control the gelation time. The ionization-control alginate hydrogels were fabricated by adding powdered calcium sulfate (Wako Pure Chemical Industries, Ltd.) as a poorly soluble calcium salt to a final concentration of 4.5% (w / v) and powdered sodium pyrophosphate (Wako Pure Chemical Industries, Ltd.) as a chelating agent to a final concentration of 0.1% (w / v) to a 4.5% (w / v) sodium alginate solution, suspending the mixture, and then allowing it to stand at room temperature for 4 hours. The alginate hydrogels with this composition gradually gelled over time from a liquid state, and could be removed from the mold once they were completely gelled.

[0059] The appearance of the molded ionization-controlled alginate hydrogel is shown in Figure 6. The ionization-controlled alginate hydrogel possessed high moldability that allowed it to precisely reflect the shape of the face, as well as high heat resistance that could withstand autoclave sterilization, flexibility, and medium water content.

[0060] From the above results, it was determined that alginate hydrogel is the optimal material for a culture scaffold, as it satisfies all the requirements of moldability, water content, heat resistance, flexibility, and cost.

[0061] (Example 3) Optimization of alginate hydrogel preparation conditions The preparation conditions for alginate hydrogel were examined in more detail. (1) Sodium alginate concentration Alginate hydrogels were prepared and evaluated in the same manner as in Example 2 using sodium alginate solutions of the concentrations shown in Table 2 below. Evaluation was based on the moldability of the scaffold and the feasibility of preparing sodium alginate, and was rated on a four-point scale of ×, △, ◯, and ⊚.

[0062] [Table 2]

[0063] As shown in Table 2, at sodium alginate concentrations below 0.5% (w / v), the hydrogel was too soft, making it difficult to mold scaffolds. At 1.0%–1.5% (w / v), scaffolds could be molded, but they were somewhat soft. At concentrations between 2.0% and 7.0% (w / v), scaffolds with a certain degree of strength could be fabricated. Of these, those fabricated at 3.0%–5.0% (w / v) were found to be the most flexible and suitable for culture. On the other hand, at concentrations between 7.5% and 8.0% (w / v), the scaffolds became too stiff and their water absorption decreased. At concentrations above 8.5% (w / v), the viscosity of the sodium alginate solution became too high, making adjustment difficult, and it was shown that it was unsuitable for mixing with poorly soluble calcium salts and chelating agents.

[0064] (2) Composition study of ionization-controlled alginate hydrogel The composition of sparingly soluble calcium salts and chelating agents in ionization-control alginate hydrogels was investigated. The sodium alginate concentration was fixed at 4.5% (w / v), and the formation of hydrogels with calcium sulfate and sodium pyrophosphate at various concentrations, as shown in Table 3, was evaluated. As in (1), evaluation was performed using a four-point scale: ×, △, ○, and ◎.

[0065] The results are shown in Table 3 below.

[0066] [Table 3]

[0067] At a concentration of 0% (w / v) of calcium sulfate, a poorly soluble calcium salt, alginate did not gel and no hydrogel was formed. At calcium sulfate concentrations of 0.5% to 1.0% (w / v), gelation took too long and the formed hydrogel was too soft, making it unsuitable for use as a culture scaffold. At concentrations above 8.0% (w / v), calcium sulfate did not disperse uniformly in the sodium alginate solution, resulting in the formation of calcium sulfate clumps on the scaffold surface, making it unsuitable. At 0% (w / v) of sodium pyrophosphate, a chelating agent, the gelation time was too rapid, making it difficult to mold a scaffold. On the other hand, at concentrations of 0.02% to 0.40% (w / v), the gelation time and hardness of the molded hydrogel were comparable. These results suggest that the most suitable composition for ionization-controlled alginate hydrogels is 0.02% (w / v) or more sodium pyrophosphate and 1.5% to 7.5% (w / v) calcium sulfate. Similar to sodium alginate, the most stable and durable hydrogels were obtained with calcium sulfate concentrations of 3.0 to 5.0% (w / v).

[0068] Based on these results, we decided to use an ionized control alginate hydrogel made with a sodium alginate concentration of 4.5% (w / v), calcium sulfate concentration of 4.5% (w / v), and sodium pyrophosphate concentration of 0.1% (w / v) as the culture scaffold for subsequent preparation of the artificial skin model.

[0069] (Example 4) Preparation of artificial skin model Next, an artificial skin model was fabricated using the collagen gel prepared in Example 1 on an ionized control alginate hydrogel scaffold. Figure 7 shows the specific flow chart for fabricating the artificial skin model. A culture scaffold reflecting the shape of the face was conditioned with differentiation medium until the epidermal stem cells reached confluence. For conditioning, the scaffold was sterilized by autoclaving and immersed in CnT-Prime 3D Barrier Culture Medium (manufactured by CELLnTEC), a commercially available keratinocyte differentiation medium, and allowed to stand (Step 1). Subsequently, the collagen gel in which the epidermal stem cells had reached confluence was mounted on the culture scaffold (Step 2), and immersion culture was performed in differentiation medium for 8 days (Step 3). The model was then exposed to the outside air for 2 days (Step 4). Photographs of the exterior of the fabricated artificial skin model were taken.

[0070] Figure 8 shows the appearance of the fabricated artificial skin. The surface appearance of the artificial skin model fabricated on alginate hydrogel was similar to that of living skin and accurately reproduced the facial skin of the subject. When the tissue was lifted from the outer edge of the alginate hydrogel with tweezers, it was observed that the entire facial scaffold was covered with a uniform sheet of skin tissue.

[0071] The skin tissue was fixed with 4% PFA, paraffin sections were prepared, and the tissue condition was confirmed by hematoxylin-eosin staining.

[0072] Figure 9 shows the results of staining tissue sections. The tissue of the artificial skin model created on alginate hydrogel was shown to have the structure of normal epidermal and dermal tissue, similar to that of living skin tissue. Furthermore, because alginate hydrogel has high water content, it was confirmed that normal skin tissue was formed uniformly over a wide area. Similar results were obtained when immortalized epidermal stem cell model cells and dermal stem cell model cells were used. These results demonstrate that the culture method using alginate hydrogel as a culture scaffold is superior for creating artificial skin models of any shape.

[0073] (Example 5) Preparation of an artificial skin model using plucked hair follicles It is known that skin barrier function, turnover rate, and sensitivity to ultraviolet light and skin irritants vary from person to person. Using the technology for producing an artificial skin model of the present invention, we attempted to create an artificial skin model that reflects the characteristics of an individual's skin. To reproduce an individual's skin, hair was removed from a living model (donor) and hair follicle tissue was harvested. The harvested hair follicle tissue was placed in a petri dish coated with iMatrix-511, immersed in Greens medium, and cultured with a cover glass mounted on top. After one week of culture, migration of hair follicle epithelial stem cells was confirmed from the bulge region of the extracted hair follicles. These cells were passaged and maintained and expanded in HuMedia-KG2 medium to obtain hair follicle epithelial stem cells.

[0074] Figure 10 shows the hair follicle epithelial stem cells that migrated from the extracted hair follicles and the culture image of the cells after subculture. Using these hair follicle epithelial stem cells, we attempted to create an artificial skin model on an alginate hydrogel scaffold that had the shape of the face of the hair follicle donor.

[0075] The appearance of the produced model is shown in Figure 11. It was confirmed that, as with epidermal stem cells, it was possible to produce an artificial skin model with a uniform and highly realistic appearance when using hair follicle epithelial stem cells.

[0076] These results demonstrate that the use of alginate hydrogel culture scaffolds makes it possible to create artificial skin models that replicate an individual's facial shape and cellular structure. Using artificial skin models that replicate an individual's skin at the cellular level allows for evaluation of skin irritation, efficacy, and UV exposure based on individual sensitivity, making them useful for the development of personalized cosmetics and drug discovery. Furthermore, stem cells derived from extracted hair follicles, which are minimally invasive to harvest, can be used to create artificial skin models tailored to the shape of the affected area. This eliminates the need for surgery, unlike conventional cell harvesting from excised skin, and allows for transplantation therapy that fits the shape of the desired area. Furthermore, it is possible to create skin coverings for humanoid robots from hair.

[0077] (Example 6) Verification of the usefulness of an artificial skin model with a facial shape (Experiment 1) Visualization of skin damage areas by UV irradiation direction Damage caused by ultraviolet light was evaluated using the artificial skin model having the shape of a face prepared in Example 4. Specifically, we investigated whether there was a change in the area of ​​skin damage that appeared depending on the angle of irradiation with ultraviolet light (UVB).

[0078] Figure 12 shows the conditions for UV irradiation. The artificial skin model with the shape of the face was placed horizontally on its back and vertically. The intensity was 30 mJ / cm. 2 The next day after irradiation, the area of ​​surviving cells was visualized using MTT staining reagent (Tokyo Chemical Industry Co., Ltd.).

[0079] Figure 13 shows the results of MTT staining. In the control (not irradiated with UVB), viable cells were observed all over the face, but when the model was placed horizontally on its back and irradiated with UVB, severe damage was observed all over the face. On the other hand, when the model was placed upright and irradiated with UVB, severe damage was observed in areas of the upper face where the three-dimensional shape stood out, such as the forehead, inner corners of the eyes, cheeks, and nose, while damage was less severe in areas that were in shadow, such as the mouth, chin, and jawline. From the above, it can be seen that by using a facial-shaped artificial skin model, it is possible to evaluate the effects of UV rays taking advantage of the three-dimensional shape of the face, and it is expected that this model will be applicable to the development of sunscreens, etc.

[0080] (Experiment 2) Visualization of UV-induced skin damage by season It is known that in real life, the amount and angle of UV rays differ between summer and winter. In this experiment, the artificial skin model prepared in Example 4 was irradiated with UV rays under conditions that simulated seasonal differences in the angle and intensity of UV rays, and the degree of UV damage for each season was investigated.

[0081] Figure 14 shows the conditions for UV irradiation. The artificial skin model was exposed to summer conditions (UV incident angle 80°, intensity 30 mJ / cm) that simulated the daytime on the summer solstice in Tokyo, Japan. 2) and winter conditions simulating the daytime on the winter solstice (ultraviolet light incident angle 35°, intensity 10 mJ / cm 2 ) and visualized the damaged areas by staining surviving cells with MTT staining. The results are shown in Figure 15. When UVB was irradiated under conditions simulating summer daytime, particularly severe damage occurred on the cheeks, nose, and forehead, but under conditions simulating winter daytime, mild damage was observed in the central part of the face. As such, the artificial skin model having the facial shape of the present invention can accurately reproduce damage caused by UVB, which changes with the seasons, demonstrating that it is possible to predict the risk of skin damage caused by UV rays in each season and propose beauty treatments appropriate for each season.

[0082] (Experiment 3) Analysis of shape change of artificial skin model due to change in gravity direction In real life, such as during sleep or during the day, the direction of gravity acting on the skin changes in various ways, and changes in the direction of gravity affect how noticeable facial sagging is and the areas where mechanostress occurs. We analyzed changes in the shape of the skin relative to the direction of gravity in an artificial skin model with the facial shape of the present invention.

[0083] As shown in Figure 16, the surface of the artificial skin model was photographed using an Antera 3D (manufactured by Nippon Ash Co., Ltd.) when it was placed horizontally on its back and when it was fixed upright using wire, and the surface shape was analyzed.

[0084] The photographed results are shown in Figure 17. The three-dimensional shape of the face was obtained using Antera 3D. The obtained three-dimensional shape was analyzed for changes in the shape of the facial line using analysis software for Antera 3D. The volume of the circle in the image was measured, and the shape change of the artificial skin model due to the direction of gravity was analyzed. The results are shown in Table 4 below.

[0085] [Table 4]

[0086] The results showed that the volume of the face line (circular part) increased when the artificial skin model was placed horizontally on its back, compared to when the model was placed vertically. This result mimics the sagging state of the face line in a living body, and it is thought that this model can be used to research sagging and develop methods to prevent aging.

[0087] (Example 7) Usefulness of humanoid robots as covering materials Currently, research in robotics is advancing rapidly worldwide, and efforts are underway to develop more humanoid robots with a more human-like feel. The appearance, texture, and tactile feel of a robot's surface are important factors in determining the robot's overall impression. The artificial skin model using the alginate hydrogel of the present invention has been shown to be very similar in appearance to real skin, and its elasticity was also evaluated. Elasticity was evaluated using a Cutometer DUAL MPA580 (manufactured by Courage+Khazaka), which can evaluate skin elasticity and flexibility. A probe was pressed against the cheek and forehead of a living human's skin and the cheek and forehead of a facial-shaped artificial skin model made using the alginate hydrogel of the present invention as a scaffold. Negative pressure was applied to the skin through the probe's opening, and the skin displacement was measured. The R7 value, which represents skin elasticity, was used as an index for evaluation.

[0088] The results are shown in Table 5. When the R7 value of the cheek of a living body was set to 1, the relative values ​​of elasticity of the forehead of a living body and the cheek and forehead of the artificial skin model were calculated.

[0089] [Table 5]

[0090] The elasticity of the created facial-shaped artificial skin model was shown to be close to that of real skin. Based on these findings, the artificial skin model using alginate hydrogel as a scaffold not only reproduces real skin in appearance but also in elasticity, and is expected to be useful as a covering material for new humanoid robots, as it also reproduces the feel and texture of real human skin.

[0091] (Example 8) Preparation of a skin-colored artificial skin model A 10% hemoglobin solution in water and a 2% β-carotene solution in DMSO were prepared, and these were added to a sodium alginate solution to a 200-fold dilution when preparing the culture scaffold. After thorough suspension, an alginate hydrogel was obtained. Then, 1 × 10 epidermal stem cells were implanted on the surface of the collagen gel. 6 At the same time as seeding the cells, 5 × 10 commercially available melanocytes (Kurabo) were seeded on the collagen gel. 4 The cells were seeded and cultured in HuMedia-KG2 medium until the epidermal stem cells reached confluence. Differentiation was then induced in the same manner to produce artificial skin. The resulting artificial skin was photographed.

[0092] Figure 18 shows the appearance of the artificial skin produced. It was demonstrated that it is possible to produce a skin-colored artificial skin model. By reproducing the color of real skin, it is expected to be applicable to coating materials for more human-like humanoid robots. In addition, a spectrophotometer CM2600d (Konica Minolta) was used to compare the color of the skin of a living human face with the color of the artificial skin model. The results are shown in Table 6.

[0093] [Table 6]

[0094] L, an index of lightness * and a, which is an index of color coordinates * and b * In this study, the artificial skin model with the skin color showed similar values ​​to those of living skin. [Industrial Applicability]

[0095] The artificial skin model of the present invention can be used in fields such as the development of cosmetics and pharmaceuticals, drug discovery research, as a transplant material in the field of regenerative medicine, and as a coating material in the development of humanoid robots.

Claims

1. An artificial skin model having an arbitrary shape, comprising a culture scaffold made of alginate hydrogel molded into an arbitrary shape, and cultured skin tissue on the culture scaffold.

2. The artificial skin model according to claim 1 , wherein the alginate hydrogel is a gel of sodium alginate with a sparingly soluble calcium salt.

3. The artificial skin model according to claim 2, wherein the poorly soluble calcium salt is calcium sulfate.

4. The artificial skin model according to claim 1, wherein the alginate hydrogel is a gel formed from a poorly soluble calcium salt of sodium alginate and a chelating agent.

5. The artificial skin model of claim 4, wherein the chelating agent is sodium pyrophosphate.

6. The artificial skin model according to claim 1, wherein the alginate hydrogel is a gel of sodium alginate with calcium sulfate and sodium pyrophosphate.

7. The artificial skin model of claim 1 , wherein the cultured skin tissue comprises epidermal tissue and dermal tissue.

8. The artificial skin model according to claim 7, wherein the cultured skin tissue is reconstructed from epidermal keratinocytes and dermal fibroblasts.

9. The artificial skin model according to claim 8, wherein the epidermal keratinocytes and fibroblasts are reconstructed from epidermal stem cells or progenitor cells and dermal stem cells or progenitor cells.

10. The artificial skin model according to claim 9 , wherein the stem or progenitor cells are immortalized stem or progenitor cells.

11. The artificial skin model of claim 9, wherein the stem cells are hair follicle-derived stem cells.

12. An artificial skin model that reproduces the color of living skin, comprising a culture scaffold made of alginate hydrogel molded into any shape and cultured skin tissue on top of the culture scaffold.

13. The artificial skin model according to claim 12, wherein the alginate hydrogel contains one or two components selected from hemoglobin and beta-carotene, which determine skin color.

14. The artificial skin model of claim 12, wherein the cultured skin tissue has epidermal tissue containing melanocytes.

15. The artificial skin model described in claim 12, characterized in that the alginate hydrogel contains one or two components selected from hemoglobin and beta-carotene, which determine skin color, and the cultured skin tissue has epidermal tissue containing melanocytes.

16. The artificial skin model of claim 1 , wherein the shape of the artificial skin model is a face shape.

17. The artificial skin model according to claim 1, wherein the artificial skin model is used for evaluation tests of the efficacy and safety of ingredients of pharmaceuticals, quasi-drugs, or cosmetics.

18. The artificial skin model of claim 1 , wherein the artificial skin model is for skin transplantation.

19. The artificial skin model of claim 1 , wherein the artificial skin model is for covering a humanoid robot.

20. A method for producing an artificial skin model, comprising the following steps: (a) A step of creating a mold with the shape of the target skin carved out using a 3D modeling machine (b) A step of gelling sodium alginate in the mold prepared in step (a) to prepare an alginate hydrogel. (c) removing the alginate hydrogel prepared in step (b) from the mold, culturing skin cells using the alginate hydrogel as a culture scaffold, and inducing differentiation to reconstruct skin tissue.

21. A method for visualizing areas of cell damage caused by ultraviolet light, comprising irradiating an artificial skin model having the facial shape described in claim 16 with ultraviolet light and staining the cells of the skin model after irradiation.

22. A method for analyzing changes in skin shape due to changes in the direction of gravity using an artificial skin model having the facial shape according to claim 16.

23. A humanoid robot that is covered with the artificial skin model according to claim 1 and has the texture and elasticity of living skin.

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