Skin model and method for producing same, and method for evaluating factors for treating or preventing skin pigmentation - Patents.com

Abstract

To provide a new skin model that can be cultured stably for a long time and a method for producing the same.SOLUTION: A skin model contains: a first cell group inoculated onto a cell culture substrate and containing fibroblasts; and a soluble component-permeable porous membrane having one face inoculated with a second cell group containing melanocytes and the other side inoculated with a third cell group containing keratinocytes, the porous membrane disposed above the first cell group. The fibroblasts are fibroblasts damaged by light irradiation. The first cell group, the second cell group and the third cell group are cocultured in the same medium without mixed. There are also provided a method for producing a skin model, and a method for evaluating a factor for treating or preventing skin pigmentation, using the skin model.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a skin model and a method for producing the same, and also to a method for evaluating factors for treating or preventing skin pigmentation using the skin model. [Background technology]

[0002] The skin is an organ covering the body surface that separates the internal and external environments. It acts as a physical barrier, protecting the body from dryness and the intrusion of harmful substances, and plays an essential role in maintaining life.

[0003] The skin of higher vertebrates is broadly divided into the epidermis, dermis, and subcutaneous tissue, starting from the outermost layer. The epidermis is primarily composed of cells called keratinocytes, which divide in the deepest part of the epidermis (basal layer) and differentiate into the spinous layer, granular layer, and stratum corneum, before migrating to the surface and eventually becoming sebum and being shed.

[0004] Melanocytes (pigment cells) exist in the basal layer of the epidermis, and melanin is produced in melanosomes within the melanocytes. The produced melanin is taken up by surrounding keratinocytes. The taken-up melanin migrates to the stratum corneum as keratinocytes turn over, and is excreted from the body over a period of approximately 40 days.

[0005] Pigmentation such as age spots and freckles on the skin is thought to be caused by factors such as hormonal abnormalities, UV exposure, and local inflammation, which can lead to excessive melanin production in melanocytes or the deposition of melanin granules in keratinocytes in the basal layer of the epidermis. Methods and therapeutic agents (skin whitening agents) for treating or preventing pigmentation, such as age-related senile lentigo, have been developed, but they often fail to produce the expected results, or only achieve temporary results without lasting effects, resulting in recurrence. As a result, there is a continuing need to develop new treatments, prevention methods, and therapeutic agents.

[0006] Under these circumstances, in order to develop new treatments, skin models that mimic the structure and function of the skin have been developed and used in recent years instead of conventional animal models (e.g., Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6113393 [Patent Document 2] International Publication No. 2020 / 111265 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a new skin model that is stable, has little variation in quality, and can be evaluated in short- to medium-long-term experimental systems (e.g., about 3 to 21 days), as well as a method for producing the same. Another object of the present invention is to provide a method for evaluating candidate factors for treating or preventing skin pigmentation using the new skin model. [Means for solving the problem]

[0009] As a result of extensive research by the present inventors, it has been found that a first cell group containing fibroblasts seeded on a cell culture substrate; It has been found that a skin model comprising: a soluble component-permeable porous membrane on one side of which a second cell group comprising melanocytes is seeded and on the other side of which a third cell group comprising keratinocytes is seeded; and a porous membrane placed above the first cell group; is stable with little variation in quality and can be used as a skin model that can be evaluated in short- to medium- to long-term experimental systems (e.g., about 3 to 21 days). That is, the present invention encompasses the following inventions.

[0010] [1] A first cell group including fibroblasts seeded on a cell culture substrate; a soluble component-permeable porous membrane having a second cell group including melanocytes seeded on one side and a third cell group including keratinocytes seeded on the other side, the porous membrane being positioned above the first cell group; A skin model comprising: the fibroblasts are fibroblasts damaged by light irradiation, A skin model in which the first cell group, the second cell group, and the third cell group are co-cultured in the same medium without being mixed. [2] The skin model according to Item 1, wherein the fibroblasts damaged by photoirradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer. [3] The skin model according to item 2, wherein the photosensitizer is selected from the group consisting of psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol-protected gold nanoclusters (AUxSRy). [4] The skin model according to any one of items 1 to 3, wherein the light irradiation is UVA light irradiation. [5] The skin model according to any one of items 1 to 4, wherein the porous membrane is made of a biocompatible component. [6] The skin model according to any one of items 1 to 5, wherein the porous membrane is a collagen membrane. [7] The skin model according to any one of items 1 to 6, wherein the first cell group further comprises fibroblasts to be evaluated.

[0011] [8] A method for producing a skin model, comprising: a step of disposing a soluble component-permeable porous membrane, on one side of which a second cell group including melanocytes is seeded and on the other side of which a third cell group including keratinocytes is seeded, above a cell culture substrate on which a first cell group including fibroblasts damaged by light irradiation is seeded, and co-culturing the first cell group, the second cell group, and the third cell group in the same medium without being mixed; A method comprising: [9] The method according to Item 8, wherein the fibroblasts damaged by photoirradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.

[10] The method of item 9, wherein the photosensitizer is selected from the group consisting of psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol-protected gold nanoclusters (AUxSRy).

[11] The method according to any one of items 8 to 10, wherein the light irradiation is UVA light irradiation.

[12] The method according to any one of items 8 to 11, wherein the porous membrane is made of a biocompatible component.

[13] The method according to any one of items 8 to 12, wherein the porous membrane is a collagen membrane.

[14] The method according to any one of items 8 to 13, wherein the first cell group further comprises fibroblasts to be evaluated.

[0012]

[15] A method for evaluating a factor for treating or preventing skin pigmentation, comprising: (1) A step of preparing a skin model by disposing a soluble component-permeable porous membrane, on one side of which a second cell group including melanocytes is seeded and on the other side of which a third cell group including keratinocytes is seeded, above a cell culture substrate on which a first cell group including fibroblasts damaged by light irradiation is seeded, and co-culturing the first cell group, the second cell group, and the third cell group in the same medium without mixing. (2) applying a candidate factor to the skin model and culturing it; (3) analyzing the pigmentation index of the first cell group obtained in step (2) and evaluating the therapeutic or preventive effect of the candidate factor; A method comprising:

[16] The method of Item 15, wherein the candidate factor is a fibroblast and is applied to the first cell group.

[17] The method according to Item 15 or 16, wherein the fibroblasts damaged by photoirradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.

[18] The method of item 17, wherein the photosensitizer is selected from the group consisting of psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol-protected gold nanoclusters (AUxSRy).

[19] The method according to any one of items 15 to 18, wherein the porous membrane is made of a biocompatible component.

[20] The method according to any one of items 15 to 19, wherein the porous membrane is a collagen membrane.

[21] The method according to any one of items 15 to 20, wherein the light irradiation is UVA light irradiation. [Effects of the Invention]

[0013] The present invention provides a skin model that is stable and has little variation in quality, and that allows confirmation of the formation of pseudo-blemish sites even in short- to medium- to long-term experimental systems (e.g., about 3 to 21 days). Furthermore, the use of the skin model provided by the present invention enables the search and evaluation of candidate factors for treating or preventing skin pigmentation. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a schematic diagram showing a method for producing a skin model according to one embodiment, with each component mainly shown in cross section. [Figure 2] Figure 2 shows the results of measuring the intracellular melanin content using the skin model in Figure 1. Top: photographs of the appearance of an equal amount of melanocyte lysate, bottom: results of measuring an equal amount of melanocyte lysate at OD 405 nm. [Figure 3] Figure 3 shows the relative expression levels (with PUVA-Fb alone set to 1) of melanin production-related genes in melanocytes when the fibroblasts to be evaluated (PVUA-untreated fibroblasts) were added using the skin model shown in Figure 1. *: p<0.05, **: p<0.01, ***: p<0.01. [Figure 4]Figure 4 shows the relative expression levels (with PUVA-Fb alone set to 1) of melanin production-related genes in keratinocytes when the fibroblasts to be evaluated (PVUA-untreated fibroblasts) were added using the skin model shown in Figure 1. *: p<0.05, **: p<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0015] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, etc. However, the technical scope of the present invention is not limited to the following embodiments.

[0016] In this specification, terms such as "first," "second," and "third" are used to distinguish one element from another; for example, a first element may be expressed as a second element, and similarly, a second element may be expressed as a first element, without departing from the scope of the present invention.

[0017] Unless otherwise defined, all terms (technical and scientific) used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] <Skin model and its manufacturing method> 1 is a schematic diagram illustrating a skin model 1 and a method for producing the same in one embodiment. In one embodiment, the skin model 1 comprises a first cell group including fibroblasts seeded on a cell culture substrate; a soluble component-permeable porous membrane having a second cell group including melanocytes seeded on one side and a third cell group including keratinocytes seeded on the other side, the porous membrane being positioned above the first cell group; A skin model comprising: the fibroblasts are fibroblasts damaged by light irradiation, This is a skin model in which the first cell group, the second cell group, and the third cell group are co-cultured in the same medium without being mixed (see, for example, Figures 1(E) and (F)).

[0019] In one embodiment, the skin model 1 is a step of disposing a soluble component-permeable porous membrane, on one side of which a second cell group including melanocytes is seeded and on the other side of which a third cell group including keratinocytes is seeded, above a cell culture substrate on which a first cell group including fibroblasts damaged by light irradiation is seeded, and co-culturing the first cell group, the second cell group, and the third cell group in the same medium without being mixed; The method can be provided by a method comprising:

[0020] Keratinocytes are one of the cells that make up the epidermis. In the epidermal tissue of a living body, they divide in the deepest part (basal layer), differentiate towards the upper layers, and then move to the surface while differentiating into the spinous layer, granular layer, and stratum corneum, before eventually becoming dandruff and falling off.

[0021] Melanocytes (pigment cells) are one of the cells that make up the epidermal tissue. In the body, they exist in the basal layer of the epidermis and produce melanin.

[0022] Fibroblasts are one of the cells that make up connective tissue and are present in many organs and tissues. In the skin, fibroblasts are mainly found in the dermis tissue. The fibroblasts used in the skin model 1 of the present invention are preferably dermis-derived fibroblasts.

[0023] The fibroblasts, keratinocytes, and melanocytes used in the present invention may each be primary cultured cells collected from biological tissue, or may be cells that have been isolated and / or grown in advance and are commercially available or distributed, or may be established cell lines, or may be cells induced to differentiate from pluripotent stem cells such as ES cells, iPS cells, or Muse cells.

[0024] The cells used in the present invention may be derived from any animal, but are preferably derived from vertebrates, more preferably from mammals, and most preferably from humans.

[0025] The fibroblasts contained in the first cell group 10 are fibroblasts that have been damaged by light irradiation. The fibroblasts that have been damaged by light irradiation are preferably damaged by light irradiation in the presence of a photosensitizer. Examples of photosensitizers that can be used include psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol-protected gold nanoclusters (AUxSRy). The use of a photosensitizer sensitizes the irradiated light, allowing for efficient damage to the fibroblasts.

[0026] The light used to damage fibroblasts may have a wavelength that damages intracellular nucleic acids, such as DNA and RNA, but does not kill all cells; ultraviolet light (approximately 200 nm to approximately 400 nm) is preferred, and UVA (approximately 320 nm to approximately 400 nm) is more preferred. The intensity of the irradiated light may be such that it damages intracellular nucleic acids, such as DNA and RNA, but does not induce apoptosis or the like, causing all cells to die; this may be adjusted appropriately depending on the wavelength, irradiation time, cell density, etc. For example, when irradiating UVA, the intensity is 0.01 J / cm. 2 ~100J / cm 2 , preferably 0.1 J / cm 2 ~20J / cm 2 , more preferably 0.5 J / cm 2 ~10J / cm 2 can be irradiated.

[0027] Culturing light-irradiated fibroblasts for a certain period of time prevents apoptosis and allows only surviving fibroblasts to proliferate (corresponding to (A) in Figure 1). Fibroblasts damaged by light irradiation exhibit characteristics of increased cellular senescence, such as elongated cell morphology, decreased proliferation, and increased production of melanin-producing factors (e.g., stem cell factor (SCF)). The level of cellular senescence can be assessed by measuring commonly known cellular senescence markers, such as the expression level of senescence-associated acid β-galactosidase (SA-βgal), constitutive activation of cell cycle control mechanisms such as the p21 / p53 pathway and p16 pathway, and the expression level of senescence-associated secretory phenotype (SASP) factors such as IL-6. By adjusting the light irradiation dose, fibroblasts with the desired senescence level can be obtained.

[0028] The fibroblasts contained in first cell group 10 constituting skin model 1 are fibroblasts that are damaged by light irradiation but survive without dying. Therefore, skin model 1 of the present invention contains roughly homogeneous fibroblasts, making it possible to provide a stable system with little variation in activity.

[0029] In one embodiment, a second cell group 20 comprising melanocytes that constitute the skin model 1 is seeded on one side of a porous membrane 22 using a second cell culture substrate 21 having a soluble component-permeable porous membrane 22 (e.g., FIG. 1(C)). After the second cell group 20 has adhered to one side of the porous membrane 22, a third cell group 30 comprising keratinocytes is seeded on the other side of the porous membrane 22 (e.g., FIG. 1(D)). The order in which the second cell group 20 and the third cell group 30 are seeded is not limited; the third cell group 30 comprising keratinocytes may be seeded first on one side of the porous membrane 22, or the second cell group 20 comprising melanocytes may be seeded first on one side of the porous membrane 22.

[0030] In the skin model 1, the porous membrane 22 is a porous membrane permeable to soluble components. It may be, for example, a cell culture insert, but is preferably made of or coated with a biocompatible component used in cell-adhesive scaffolds. For example, the biocompatible component may be made of or coated with a material selected from the group consisting of collagen, gelatin, hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, fibrinogen / thrombin, fibrillin, elastin, gum, cellulose, agar, gluten, casein, albumin, vitronectin, tenascin, entactin / nidogen, glycoproteins, glycosaminoglycans, poly(acrylic acid) and its derivatives, poly(ethylene oxide) and its copolymers, poly(vinyl alcohol), polyphosphazene, Matrigel, and combinations thereof. Furthermore, the porous membrane 22 is more preferably a collagen membrane. The second cell culture substrate 21, in which the porous membrane 22 is a collagen membrane, can be a known material, for example, ad-MED Vitrigel from Kanto Chemical Co., Ltd. (Japan) or a permeable collagen membrane from Koken Co., Ltd. (Japan).

[0031] The average pore size of the porous membrane 22 may be, for example, about 0.01 μm to about 100 μm (for example, 0.01 μm to 100 μm, 0.01 μm to 50 μm, 0.01 μm to 10 μm, 0.1 μm to 50 μm, or 0.1 μm to 10 μm). The pore density of the porous membrane 22 can also be appropriately selected, but for example, 1×10 4 / cm 2 That's it, 1 x 10 5 / cm 2 That's it, 5 x 10 5 / cm 2 That's it, 10 x 10 5 / cm 2 That's it, 50 x 10 5 / cm 2 or more, or 100 x 10 5 / cm 2 The pore density may be greater than or equal to 1 x 10 4 / cm2 ~100×10 8 / cm 2 , 1×10 5 / cm 2 ~100×10 8 / cm 2 may be.

[0032] In one embodiment, the first, second, or third cell group used in the skin model 1 of the present invention may contain cells other than those described above, such as Langerhans cells or Merkel cells, which are contained in the epidermis. The number of cells seeded in the skin model 1 is not particularly limited, but may be, for example, 1 × 10 2 ~10 6 pieces / cm 2 , 1.0 to 10 × 10 4 pieces / cm 2 , or approximately 4 to 8 × 10 4 pieces / cm 2 It may also include.

[0033] In the skin model 1 of the present invention, a second cell culture substrate 21, in which a second cell group 20 containing melanocytes and a third cell group 30 containing keratinocytes are seeded on both sides of a porous membrane 22, is placed above a first cell group 10. The first cell group 10, the second cell group 20, and the third cell group 30 are co-cultured in the same medium without being mixed with each other. This allows the soluble components produced by the first cell group 10, the second cell group 20, and the third cell group 30 to be exchanged.

[0034] In one embodiment, the cells used in the present invention may be, for example, cells contained in commercially available products such as TESTSKIN™ LSE-melano (TOYOBO) and MelanoDerm™ (MatTek).

[0035] The skin model 1 can be cultured for 0 to 14 days at approximately 37° C. using a culture medium typically used for keratinocyte culture, such as KG medium, Epilife KG2 (Kurabo Industries, Ltd.), Humedia-KG2 (Kurabo Industries, Ltd.), or assay medium (Toyobo, Ltd.). Other culture media that can be used include DMEM medium (GIBCO) or a 1:1 mixture of ascorbic acid-containing KGM and DMEM.

[0036] In one embodiment, the culture medium for the skin model 1 may contain ascorbic acid, an ascorbic acid derivative, or a salt thereof. The presence of ascorbic acid, an ascorbic acid derivative, or a salt thereof promotes fibroblast proliferation and collagen production, thereby promoting stratification similar to the structure of the dermis. As used herein, "ascorbic acid derivative" refers to, for example, ascorbic acid 2-phosphate, ascorbic acid 1-phosphate, sodium L-ascorbate, L-ascorbic acid 2-glucoside, etc., and also includes salts thereof (sodium salt, magnesium salt, etc.).

[0037] The skin model 1 provided by the present invention can be used to evaluate various factors that cause skin pigmentation, such as the direct or indirect action of fibroblasts damaged by light irradiation, such as keratinocyte hyperproliferation, hyperdifferentiation, or pigment granule uptake, and melanin production by melanocytes. Furthermore, by measuring and analyzing "pigmentation indicators" in the skin model 1, factors that affect pigmentation can also be evaluated.

[0038] By using the skin model 1 of the present invention, it becomes possible to evaluate, for example, candidate factors to be evaluated for treating or preventing skin pigmentation.

[0039] <Method for evaluating factors for treating or preventing skin pigmentation> The present invention can provide a method for evaluating a factor for treating or preventing skin pigmentation using a skin model. In one embodiment, the method of the present invention comprises: (1) A step of preparing a skin model by disposing a soluble component-permeable porous membrane, on one side of which a second cell group including melanocytes is seeded and on the other side of which a third cell group including keratinocytes is seeded, above a cell culture substrate on which a first cell group including fibroblasts damaged by light irradiation is seeded, and co-culturing the first cell group, the second cell group, and the third cell group in the same medium without mixing. (2) applying a candidate factor to the skin model and culturing it; (3) analyzing the pigmentation index of the first cell group obtained in step (2) and evaluating the therapeutic or preventive effect of the candidate factor; Includes.

[0040] In one embodiment, the candidate factor may be, for example, a small molecule compound, a peptide, a nucleic acid, a protein, a mammalian cell (e.g., mouse, rat, pig, cow, sheep, monkey, human, etc.), a tissue extract or cell culture supernatant, a plant-derived compound or extract (e.g., herbal extract, a compound derived from a herbal drug), and a microbial compound, extract, or culture product.

[0041] In one embodiment, the candidate factor CA may be any cell, such as a fibroblast precursor cell including a mesenchymal stem cell, or a fibroblast (e.g., a fibroblast not damaged by light irradiation or a fibroblast less damaged by light irradiation), as shown in Figure 1(F). As used herein, "fibroblasts not damaged by light irradiation" or "fibroblasts less damaged by light irradiation" refer to fibroblasts that have not been subjected to the above-mentioned light irradiation step and have a lower level of senescence than the above-mentioned "fibroblasts damaged by light irradiation."

[0042] The candidate substance can be added to the skin model 1, and after culturing for a desired period of time, the pigmentation therapeutic or preventive effect of the candidate substance can be evaluated by analyzing the pigmentation index in the skin model 1. For example, if the pigmentation index in the skin model 1 to which the candidate substance is added is improved by comparing it with the pigment production and / or pigmentation level in the skin model 1 to which the candidate substance is not added or to which any substance that does not have a pigmentation therapeutic or preventive effect is added, the candidate substance can be evaluated as having a pigmentation therapeutic or preventive effect.

[0043] Examples of "pigmentation indicators" include, but are not limited to, differences in the amount of pigment contained, turnover levels (e.g., changes in the expression of differentiation markers), aggregation or diffusion of pigment granules, heterogeneity of cytoplasm, expression of melanin synthesis factors (e.g., tyrosinase (TYR); tyrosinase-related protein 1 (TYRP1); dopachrome tautomerase (DCT); or microphthalmia-associated transcription factor (MITF) in melanocytes), or endothelin 1 (ET1); or stem cell factor (SCF1) in keratinocytes), increased differentiation or proliferation ability (e.g., Ki67), differential expression of pigment granule distribution, or increased phagocytosis of keratinocytes.

[0044] As used herein, "pigment production" refers to the production of pigments, such as melanin, produced by melanocytes when the skin model 1 contains melanocytes. The amount of pigment produced can be determined, for example, by extracting melanin from the skin model, particularly the second cell group 20, and measuring the absorbance at 405 nm. The amount of pigment produced can also be measured, for example, by measuring the amount of melanin or the amount of nucleic acid (e.g., mRNA amount) encoding it contained in the skin model, particularly the second cell group 20, using methods such as, but not limited to, ELISA, flow cytometry, Western blotting, immunohistochemistry, and qPCR.

[0045] As used herein, "degree of pigmentation" refers to the color brightness of the skin model, particularly the first cell group, under visible light. The brightness of the skin model 1 of the present invention varies depending on the amount of melanin produced by melanocytes. Therefore, as the amount of melanin increases, the brightness decreases, and the second cell group 20 of the skin model 1 appears dark. Conversely, as the amount of melanin decreases, the brightness increases, and the second cell group 20 of the skin model 1 appears light. In other words, by comparing the color brightness of the skin model 1, the therapeutic or preventive effect of the added candidate substance on pigmentation can be evaluated. Brightness can be quantified by recording an image of the second cell group 20 of the skin model 1 and using a known image measurement method. [Example]

[0046] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the present invention in any way.

[0047] 1. Materials and Experimental Methods

[0048] 1-1. Cultivation of epidermal cells

[0049] Normal human epidermal melanocytes (Kurabo) were cultured in Medium 254 (Thermo Fisher Scientific) supplemented with human melanocyte growth supplement (HMGS2). Normal human epidermal keratinocytes (Kurabo) were cultured in Epilife® Medium (Thermo Fisher Scientific) containing 60 μM calcium and supplemented with keratinocyte growth supplement (EDGS).

[0050] 1-2. Fibroblast culture and PUVA treatment Normal human fibroblasts (Cell Research Corp) were cultured in growth medium (Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum (hereinafter referred to as 10% DMEM)) and plated at 1x10 cells per well in a 6-well plate. 5Before the cell density reached 100% confluence, the cells were replaced with 10% DMEM supplemented with the photosensitizer psoralen (final concentration 25 ng / mL) and cultured. After 24 hours, the cells were swollen with 1 mL of PBS, removed, and replaced with 1 mL of PBS supplemented with psoralen (final concentration 25 ng / mL), and then cultured at 6 J / cm. 2 The cells were then irradiated with UVA (hereafter referred to as PUVA treatment). After swelling with 1 mL of PBS, the PBS was removed and replaced with 10% DMEM. The cells were then cultured for 3 days, and the culture supernatant and cells were collected. PUVA-untreated fibroblasts from the same donor served as a control for the PUVA-treated fibroblasts.

[0051] 1-3. Preparation of a non-contact three-layer culture model of keratinocytes, pigment cells, and fibroblasts

[0052] To create the three-layer culture model, a collagen membrane capable of double-sided culture, such as ad-MED Vitrigel (Kanto Chemical Co., Ltd.) or a permeable collagen membrane (Koken Co., Ltd.), was used. Melanocytes were cultured on one side of the collagen membrane at 1 × 10 4 ~1×10 5 The cells were seeded on the other surface and cultured for 24 hours to allow them to adhere to the membrane. Then, 1 × 10 keratinocytes were seeded on the other surface. 4 cells ~ 1 × 10 5 1x10 fibroblasts pre-treated with PUVA were seeded. 4 A collagen membrane with keratinocytes and melanocytes attached was placed on a 24-well plate seeded with 100 cells / well, and co-cultured for 3–5 days to induce changes in the epidermal cells resembling pigmented spots. After induction of pigmented spot-like epidermal cells, 1 / 10, 1 / 5, 1 / 2, or an equal amount of therapeutic fibroblasts (corresponding to "non-PUVA-FB" in Figure 1(F)) were added to the 24-well plate and cultured for an additional 3–5 days to examine the therapeutic effect of fibroblasts on pigmented spot-like epidermal cells. Cultures were performed in a 1:1:1 mixture of 10% DMEM (fibroblast culture medium), M254 (melanocyte culture medium), and EpiLife (keratinocyte culture medium).

[0053] 1-4. Measurement of intracellular and extracellular melanin content

[0054] Melanocytes were placed on one side of the collagen gel at 1 × 10 5 Cells were seeded at 0.1 μg / well and cultured using the model described above. The medium was collected and centrifuged (4°C, 200 × g, 10 minutes) to remove cell debris. The resulting supernatant was measured at OD405nm and used as the amount of extracellular melanin. Melanocytes were collected by trypsinization and centrifuged (4°C, 2,300 × g, 5 minutes). 120 μl of 0.2 N NaOH was added to the resulting cell mass and the mixture was incubated in a water bath at 80°C for 30 minutes to extract intracellular melanin. The resulting supernatant was measured at OD405nm and used as the amount of intracellular melanin. Results were obtained from three independent experiments (Figure 2).

[0055] 1-5.RT-PCR Total RNA was extracted using the RNeasy Mini Kit (Qiagen). 200 ng of total RNA was reverse transcribed using the PrimeScript RT Reagent Kit (Takara). PCR was performed using the PrimeScript RT-PCR Kit (Takara). After fibroblast treatment, gene expression of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT), and microphthalmia-associated transcription factor (MITF) in melanocytes, and endothelin 1 (ET1), stem cell factor (SCF), and GAPDH in keratinocytes were analyzed by RT-PCR. PCR results were obtained from three independent experiments (Figures 3 and 4). The following primer sequences were used in this experiment: [Table 1]

[0056] 1-6.Statistical processing

[0057] Statistical analysis was performed using Graph Pad Prism 5 (Graph Pad Software, La Jolla, CA) by one-way analysis of variance. A p < 0.05 was considered statistically significant, and the degree of significance is indicated by the following asterisks: *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were repeated at least three times to confirm reproducibility.

[0058] 2.Results

[0059] Melanocytes and keratinocytes were isolated from the skin model, and intracellular melanin levels and gene expression levels related to melanin synthesis were measured. The increase in melanin levels was suppressed in the non-irradiated Fb-treated model compared with the PUVA Fb-treated model. RT-PCR analysis demonstrated that this suppression of melanin darkening was due to a decrease in gene expression of the melanin synthesis enzyme tyrosinase, tyrosinase-related genes TYRP1 and DCT, and their transcription factor MITF. Furthermore, in keratinocytes, the expression of SCF and ET1, which induce melanin synthesis, was decreased in a concentration-dependent manner. These results suggest that non-irradiated Fb acts on both melanocytes and keratinocytes to suppress PUVA Fb-induced melanin synthesis. [Explanation of symbols]

[0060] 1, 1a, 1b Skin Model 10 1st cell group 11 First cell culture substrate 12 First medium 20 Second cell group 21 Second cell culture substrate 22 Porous membrane 23 Second medium 24 Third cell culture substrate 30 Third cell group RD light irradiation FB fibroblasts bFB fibroblasts damaged by light irradiation KC keratinocytes MC melanocytes CA candidate substance

Claims

1. a first cell population comprising fibroblasts seeded on a cell culture substrate; a soluble component-permeable porous membrane having a second cell group including melanocytes seeded on one surface thereof and a third cell group including keratinocytes seeded on the other surface thereof, the porous membrane being disposed above the first cell group; A skin model comprising: the fibroblasts are fibroblasts damaged by light irradiation, A skin model, wherein the first cell group, the second cell group, and the third cell group are co-cultured in the same medium without being mixed, A skin model for confirming pseudo-spot sites formed by fibroblasts damaged by the light irradiation.

2. The skin model according to claim 1 , wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.

3. 3. The skin model of claim 2, wherein the photosensitizer is selected from the group consisting of psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol-protected gold nanoclusters (AUxSRy).

4. The skin model according to any one of claims 1 to 3, wherein the light irradiation is UVA light irradiation.

5. The skin model according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane made of a biocompatible component.

6. The skin model according to any one of claims 1 to 5, wherein the porous membrane is a collagen membrane.

7. The skin model according to any one of claims 1 to 6, wherein the first cell group further comprises fibroblasts to be evaluated.

8. A method for producing a skin model for confirming pseudo-blemish sites formed by fibroblasts damaged by light irradiation, comprising: a step of disposing a soluble component-permeable porous membrane, on one side of which a second cell group including melanocytes is seeded and on the other side of which a third cell group including keratinocytes is seeded, above a cell culture substrate on which a first cell group including fibroblasts damaged by light irradiation is seeded, and co-culturing the first cell group, the second cell group, and the third cell group in the same medium without being mixed; A method comprising:

9. The method according to claim 8, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.

10. 10. The method of claim 9, wherein the photosensitizer is selected from the group consisting of psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol-protected gold nanoclusters (AUxSRy).

11. The method according to any one of claims 8 to 10, wherein the light irradiation is UVA light irradiation.

12. The method according to any one of claims 8 to 11, wherein the porous membrane is made of a biocompatible component.

13. The method according to any one of claims 8 to 12, wherein the porous membrane is a collagen membrane.

14. The method according to any one of claims 8 to 13, wherein the first cell group further comprises fibroblasts to be evaluated.

15. 1. A method for evaluating a factor for treating or preventing skin pigmentation, comprising: (1) A step of preparing a skin model for confirming pseudo-blemish sites formed by fibroblasts damaged by light irradiation, by disposing a soluble component-permeable porous membrane, on one side of which a second cell group including melanocytes is seeded and on the other side of which a third cell group including keratinocytes is seeded, above a cell culture substrate on which a first cell group including fibroblasts damaged by light irradiation is seeded, and co-culturing the first cell group, the second cell group, and the third cell group in the same medium without mixing. (2) applying a candidate factor to the skin model and culturing it; (3) analyzing the pigmentation index of the first cell group obtained in the step (2) and evaluating the therapeutic or preventive effect of the candidate factor; A method comprising:

16. 16. The method of claim 15, wherein the candidate agent is a fibroblast and is applied to the first cell population.

17. The method according to claim 15 or 16, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.

18. 18. The method of claim 17, wherein the photosensitizer is selected from the group consisting of psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol-protected gold nanoclusters (AUxSRy).

19. The method according to any one of claims 15 to 18, wherein the porous membrane is made of a biocompatible component.

20. The method according to any one of claims 15 to 19, wherein the porous membrane is a collagen membrane.

21. The method according to any one of claims 15 to 20, wherein the light irradiation is UVA light irradiation.

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