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 first cell culture substrate and containing keratinocytes and / or melanocytes; and a second cell group that is inoculated onto a second cell culture substrate disposed above the first cell group and having a porous membrane and contains fibroblasts. 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 that covers the body surface and 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 roughly divided into the epidermis, dermis, and subcutaneous tissue layers, starting from the outermost layer. The epidermis is mainly 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 as they move upwards to the surface, where they eventually become sebum and are shed.

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

[0005] Pigmentation such as skin spots and freckles is thought to be caused by excessive melanin formation in melanocytes due to hormonal abnormalities, exposure to ultraviolet light, local inflammation, etc., or by the deposition of melanin granules in keratinocytes in the basal layer of the epidermis. Methods and treatments (skin whitening agents) for treating or preventing pigmentation, such as age-related senile lentigo, have been developed, but there are problems such as not achieving the expected effect, or only a temporary effect being achieved, which does not last and leads to recurrence, and there is a continuing need to develop new treatments or prevention methods and treatments.

[0006] Under these circumstances, in order to develop new treatments, skin models that mimic the structure and functions of the skin have been developed and used in recent years as an alternative to 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 and has little variation in quality and can be evaluated in short- to medium-long-term experimental systems (e.g., about 3 to 21 days), and 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 intensive research by the present inventors, it has been found that a first cell group including keratinocytes and / or melanocytes seeded on a first cell culture substrate; and a second cell group, which is placed above the first cell group and contains fibroblasts seeded on a second cell culture substrate having a porous membrane; the present invention has been found to be a skin model that is stable and has little variation in quality, and 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 population of cells comprising keratinocytes and / or melanocytes seeded on a first cell culture substrate; a second cell group, the second cell group including fibroblasts seeded on a second cell culture substrate having a porous membrane, the second cell group being disposed above the first cell group; A skin model comprising: The fibroblasts are fibroblasts damaged by light irradiation, Soluble components are exchanged between a first medium for culturing the first cell group and a second medium for culturing the second cell group via the porous membrane. Skin model. [2] The skin model described in 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 second cell group further comprises fibroblasts to be evaluated. [6] The skin model according to any one of items 1 to 5, wherein a substance for evaluating dye uptake is added to the first cell group.

[0011] [7] A method for producing a skin model, comprising: A step of disposing a second cell culture substrate having a porous membrane on which a second cell group including fibroblasts damaged by light irradiation is seeded above a first cell culture substrate on which a first cell group including keratinocytes and / or melanocytes is seeded, and co-culturing the first cell group and the second cell group, A process in which soluble components are exchanged between a first medium for culturing the first cell group and a second medium for culturing the second cell group through the porous membrane. A method comprising: [8] The method according to Item 7, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer. [9] The method of claim 8, 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).

[10] The method according to any one of items 7 to 9, wherein the light irradiation is UVA light irradiation.

[11] The method according to any one of items 7 to 10, wherein the second cell group further comprises fibroblasts to be evaluated.

[12] The method according to any one of items 7 to 11, wherein a substance for evaluating dye uptake is added to the first cell group.

[0012]

[13] A method for evaluating a factor for treating or preventing skin pigmentation, comprising: (1) A step of preparing a skin model by disposing a second cell culture substrate having a porous membrane on which a second cell group including fibroblasts damaged by light irradiation is seeded above a first cell culture substrate on which a first cell group including keratinocytes and / or melanocytes is seeded, and co-culturing the first cell group and the second cell group, a step of exchanging soluble components between a first medium for culturing the first cell group and a second medium for culturing the second cell group through the porous membrane; (2) applying a candidate factor to the skin model and culturing it; (3) analyzing an index of pigmentation of the first cell group obtained in the step (2) and evaluating the therapeutic or preventive effect of the candidate factor; A method comprising:

[14] The method according to Item 13, further comprising a step of adding a dye uptake evaluation substance to the first cell group prior to the step (1), and in the step (3), the degree of uptake of the dye uptake evaluation substance into the first cell group is used as an index of the pigmentation.

[15] The method according to Item 13, further comprising the step of adding a dye uptake evaluation substance to the first cell group after the step (2), and in the step (3), the degree of uptake of the dye uptake evaluation substance into the first cell group is used as an index of the pigmentation.

[16] The method according to any one of items 13 to 15, wherein the candidate factor is a fibroblast and is applied to the second cell group.

[17] The method according to any one of items 13 to 16, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.

[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 items 13 to 18, wherein the light irradiation is UVA light irradiation. Effect of the Invention

[0013] The present invention provides a skin model that is stable and has little variation in quality, and 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, by using the skin model provided by the present invention, it becomes possible to search for and evaluate candidate factors for treating or preventing skin pigmentation. [Brief description 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. [Diagram 2] 2 is a schematic diagram showing a method for producing a skin model according to one embodiment, with each component mainly shown in cross section. [Diagram 3] 4 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 4]Figure 4 shows the results of investigating the inhibitory effect on keratinocyte proliferation and differentiation when PVUA-untreated fibroblasts were added, using the skin model in Figure 1. (A) Fluorescence microscopy image of keratinocytes fluorescently stained for the proliferation marker Ki67 (green) and nuclei (blue). (B) Fluorescence microscopy image of keratinocytes fluorescently stained for the differentiation marker p63 (green) and nuclei (blue). The numbers in the bottom right of each image indicate the percentage of positive cells for each marker. [Diagram 5] Figure 5 shows the results of investigating the inhibitory effect on keratinocyte proliferation and differentiation when PVUA-untreated fibroblasts were added, using the skin model in Figure 1. Gene expression of Ki67 (A) or P63 (B) in keratinocytes after the addition of PVUA-untreated fibroblasts was analyzed by RT-PCR assay. Gene expression of GAPDH was measured as an internal standard and normalized. Each graph shows the relative value when the expression level of each gene in keratinocytes not co-cultured with PVUA-untreated fibroblasts (corresponding to "PVUA") was set to 1. *: p<0.05, **: p<0.01. [Figure 6] Figure 6 shows the results of investigating the uptake of 0.5 μm beads or melanosomes by keratinocytes when the fibroblasts to be evaluated (non-PUVA-Fb) were added using the skin model of Figure 2. (A) Fluorescence microscopy images showing the positions of 0.5 μm beads (red) and nuclei (blue) in keratinocytes. (B) Fontana-Masson staining showing the state of melanosome uptake in keratinocytes. [Figure 7] Figure 7 shows the results of examining the difference in distribution of pigment granules (melanosomes) by keratinocytes when the fibroblasts to be evaluated (non-PUVA-Fb) were added using the skin model of Figure 3. The state of melanosome uptake in keratinocytes was observed by Fontana-Masson staining. [Figure 8] Figure 8 shows the results of investigating keratinocyte aging markers when the fibroblasts to be evaluated (non-PUVA-Fb) were added using the skin model of Figure 3. Keratinocytes fluorescently stained for β-gal (green) and nuclei (blue) were observed under a fluorescent microscope. [Figure 9] Figure 9 shows the results of investigating keratinocyte aging markers when the fibroblasts to be evaluated (non-PUVA-Fb) were added using the skin model of Figure 3. The gene expression of p16 (A) or p21 (B) in keratinocytes after the addition of PVUA-untreated fibroblasts was analyzed by RT-PCR assay. Gene expression of GAPDH was measured as an internal standard and normalized. Each graph shows the relative value when the expression level of each gene in keratinocytes not co-cultured with PVUA-untreated fibroblasts (corresponding to "PVUA") was set to 1. *: p<0.05, **: p<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings, but the technical scope of the present invention is not limited to the embodiments described below.

[0016] In this specification, terms such as "first" and "second" are used to distinguish one element from another element. 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> FIG. 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 is a first population of cells comprising keratinocytes and / or melanocytes seeded onto a first cell culture substrate; a second cell group, the second cell group including fibroblasts seeded on a second cell culture substrate having a porous membrane, the second cell group being disposed above the first cell group; A skin model comprising: The fibroblasts are fibroblasts damaged by light irradiation, This is a skin model in which soluble components are exchanged between a first culture medium for culturing the first cell group and a second culture medium for culturing the second cell group via the porous membrane (see, for example, Figure 1(C)).

[0019] In one embodiment, the skin model 1 is A step of disposing a second cell culture substrate having a porous membrane on which a second cell group including fibroblasts damaged by light irradiation is seeded above a first cell culture substrate on which a first cell group including keratinocytes and / or melanocytes is seeded, and co-culturing the first cell group and the second cell group, The present invention can be provided by a method including a step of exchanging soluble components between a first culture medium for culturing the first cell group and a second culture medium for culturing the second cell group via the porous membrane.

[0020] Keratinocytes are one of the cells that make up the epidermis. In the epidermal tissue of living organisms, they divide in the deepest part (the basal layer), and then move upwards while differentiating into the spinous layer, the granular layer, and then the stratum corneum, before moving to the surface and eventually becoming dandruff and being shed.

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

[0022] 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.

[0023] In the skin model 1, a first cell group 10 including keratinocytes and / or melanocytes is seeded on a first cell culture substrate 11. The first cell culture substrate 11 may be any known culture substrate and is not particularly limited.

[0024] In one embodiment, the first cell group 10 containing keratinocytes and / or melanocytes used in the skin model 1 of the present invention may contain cells other than keratinocytes and melanocytes, for example, Langerhans cells and Merkel cells contained in the epidermis. The first cell group 10 may contain, for example, only keratinocytes, only melanocytes, or keratinocytes and melanocytes. When both are contained, the first cell group 10 may contain cells at a ratio of keratinocytes:melanocytes of 1:1 to 1000:1, preferably 3:1 to 30:1. The number of cells seeded in the first cell group 10 is not particularly limited, but may be, for example, 1×10 2 ~10 6 pieces / cm 2 , preferably 1.0 to 10×10 4 pieces / cm 2 , more preferably about 4 to 8 × 10 4 pieces / cm 2 It may include.

[0025] In the skin model 1 of the present invention, a second cell group 20 including fibroblasts seeded on a second cell culture substrate 21 having a porous membrane 22 is disposed above a first cell group 10. A space in which a culture medium exists is provided between the second cell group 20 and the first cell group 10. Thus, soluble components are exchanged between a first culture medium 12 for culturing the first cell group 10 and a second culture medium 23 for culturing the second cell group 20 via the porous membrane 22.

[0026] In the skin model 1, the second cell culture substrate 21 having a porous membrane 22 on which the second cell group 20 is seeded can be a known culture substrate capable of culturing fibroblasts, but is preferably, for example, a cell culture insert.

[0027] The average pore size of the porous membrane 22 can be appropriately selected, and 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 density of the pores of the porous membrane 22 can also be appropriately selected, and may be, for example, 1×10 4 / cm 2 That's it, 1×10 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 / cm 2 ~100×10 8 / cm 2 , 1×10 5 / cm 2 ~100×10 8 / cm 2 may be also possible.

[0028] 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.

[0029] 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 contained in the dermis tissue. The fibroblasts used in the skin model 1 of the present invention are preferably dermis-derived fibroblasts.

[0030] The fibroblasts contained in the second cell group 20 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 gold nanoclusters protected with thiol groups (AUxSRy). By using a photosensitizer, the irradiated light is sensitized, and it is possible to efficiently damage the fibroblasts.

[0031] 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, and is preferably ultraviolet light (about 200 nm to about 400 nm), and more preferably UVA (about 320 nm to about 400 nm). 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 and kill all cells, and may be appropriately adjusted 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.

[0032] By culturing light-irradiated fibroblasts for a certain period of time, apoptosis and the like are not induced, and only surviving fibroblasts can be proliferated (corresponding to (B) in Figure 1). Fibroblasts damaged by light irradiation show characteristics of an increased level of cellular senescence, such as elongated cell morphology, decreased proliferation ability, and increased production of melanin-producing factors (e.g., stem cell factor (SCF)). The level of cellular senescence can be examined by measuring commonly known cellular senescence markers, such as the expression level of senescence-associated acid β-galactosidase (SA-βgal), the constant activation of cell cycle check mechanisms such as the p21 / p53 pathway and the p16 pathway, and the expression level of senescence-associated secretory phenotype (SASP) factors such as IL-6. Fibroblasts with a desired level of senescence can be obtained by adjusting the amount of light irradiation.

[0033] The fibroblasts contained in the second cell group 20 constituting the skin model 1 are fibroblasts that are damaged by light irradiation but survive without dying. Therefore, the skin model 1 of the present invention contains roughly homogeneous fibroblasts, and a stable system with little variation in activity can be provided.

[0034] In one embodiment, the cells contained in the first cell group 10 may be, for example, cells contained in commercially available products such as TESTSKIN (trademark) LSE-melano (TOYOBO) and MelanoDerm (trademark) (MatTek), such as keratinocytes.

[0035] The skin model 1 can be cultured for 0 to 14 days at about 37° C. using, for example, a culture medium used for normal keratinocyte culture, such as KG medium, EpilifeKG2 (Kurabo Industries, Ltd.), Humedia-KG2 (Kurabo Industries, Ltd.), assay medium (TOYOBO), etc. As the culture medium, other media that can be used include DMEM medium (GIBCO) or a 1:1 mixture of ascorbic acid-containing KGM and DMEM, etc.

[0036] In one embodiment, the medium of the skin model 1 may contain ascorbic acid, an ascorbic acid derivative or a salt thereof. When ascorbic acid, an ascorbic acid derivative or a salt thereof is present, the proliferation of fibroblasts and collagen production are promoted, thereby promoting stratification like the structure of the dermis. In this specification, the term "ascorbic acid derivative" refers to, for example, ascorbic acid diphosphate, ascorbic acid monophosphate, sodium L-ascorbate, L-ascorbic acid 2-glucoside, etc., and further includes salts thereof (sodium salt, magnesium salt, etc.).

[0037] In another embodiment, the skin model 1 of the present invention may further include a pigment uptake evaluation substance added to the first cell group 10 (e.g., 30a in FIG. 2(F) or 30b in FIG. 3(A)). The pigment uptake evaluation substance may be added after the first cell group 10 including keratinocytes and / or melanocytes is seeded on the first cell culture substrate 11 (e.g., FIG. 3(A)), or may be added later to the first cell culture substrate 11 on which the first cell group 10 has been seeded after the skin model 1 is produced (e.g., FIG. 2(F)).

[0038] The dye uptake evaluation substance applicable to the present invention may be, for example, beads or pigment granules such as melanosomes. The material of the beads is not particularly limited as long as it is used for evaluating the uptake of pigment granules, and for example, commercially available fluorescent beads made of polystyrene or latex may be used. The size of the beads may be any size that mimics pigment granules, such as melanosomes, and may be, for example, 0.01 μm to 10 μm, preferably 0.1 μm to 5 μm, and more preferably 0.5 μm to 2 μm (for example, about 1 μm). This makes it possible to evaluate the uptake action (phagocytosis) of the beads by the first cell group.

[0039] The pigment uptake evaluation substance applicable in the present invention, for example, melanosomes, can be prepared from melanocytes. For example, it may be secreted into the culture supernatant of melanocytes, and can be prepared by homogenizing melanocytes and recovering a fraction rich in melanosomes. The melanosomes recovered by these methods can be applied to the first cell group to evaluate the uptake action (phagocytosis) of melanosomes.

[0040] The skin model 1 provided by the present invention can evaluate various factors that directly or indirectly act on fibroblasts damaged by light irradiation to form skin pigmentation, such as keratinocyte hyperproliferation, hyperdifferentiation, or uptake of a pigment uptake evaluation substance, melanin production by melanocytes, etc. In addition, by measuring and analyzing "pigmentation indexes" in the skin model 1, factors that affect pigmentation can also be evaluated.

[0041] By using the skin model 1 of the present invention, it becomes possible to evaluate, for example, candidate factors as evaluation targets for treating or preventing skin pigmentation.

[0042] <Method of 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 second cell culture substrate having a porous membrane on which a second cell group including fibroblasts damaged by light irradiation is seeded above a first cell culture substrate on which a first cell group including keratinocytes and / or melanocytes is seeded, and co-culturing the first cell group and the second cell group, a step of exchanging soluble components between a first medium for culturing the first cell group and a second medium for culturing the second cell group through the porous membrane; (2) applying a candidate factor to the skin model and culturing it; (3) analyzing an index of pigmentation of the first cell group obtained in the step (2) and evaluating the therapeutic or preventive effect of the candidate factor; Contains:

[0043] 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 a cell culture supernatant, a compound or extract derived from a plant (e.g., a herbal extract, a compound derived from a herbal drug), and a compound or extract derived from a microorganism, or a culture product.

[0044] In one embodiment, the candidate factor CA may be any cell, for example, a precursor cell of a fibroblast including a mesenchymal stem cell, a fibroblast (for example, a fibroblast not damaged by light irradiation, or a fibroblast having low damage by light irradiation), etc., as shown in Figure 1 (D), Figure 2 (D), and Figure 3 (D). As used herein, "fibroblast not damaged by light irradiation" or "fibroblast having low damage by light irradiation" refers to a fibroblast that has not been subjected to the above-mentioned light irradiation step, and has a lower level of senescence than the above-mentioned "fibroblast damaged by light irradiation."

[0045] The candidate substance can be added to the skin model 1, and after culturing for a desired period of time, the pigmentation treatment or prevention effect of the candidate substance can be evaluated by analyzing the pigmentation index in the skin model 1. For example, when the pigmentation index in the skin model 1 to which the candidate substance is added is improved by comparing the degree of pigment production and / or pigmentation in the skin model 1 to which the candidate substance is not added or to which any substance having no pigmentation treatment or prevention effect is added, the candidate substance can be evaluated as having a pigmentation treatment or prevention effect.

[0046] Examples of "indicators of pigmentation" 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 uptake evaluation substances, heterogeneity of cellular traits, 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), enhancement of differentiation or proliferation ability (e.g., Ki67, etc.), differential expression of pigment granule distribution, or enhanced phagocytosis of keratinocytes.

[0047] As used herein, "pigment production" refers to the production of a pigment, 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 first cell group (keratinocytes and melanocytes), and measuring the absorbance at 405 nm. The amount of pigment produced can be determined, for example, by measuring the amount of melanin or the amount of nucleic acid (e.g., mRNA amount) encoding melanin contained in the skin model, particularly the first cell group (keratinocytes and melanocytes), using, but not limited to, ELISA, flow cytometer, Western blot, immunohistochemistry, qPCR, or other methods.

[0048] In this specification, the "degree of pigmentation" refers to the brightness of the color of the skin model, particularly the first cell group, under visible light. When the skin model 1 of the present invention contains melanocytes, the brightness changes depending on the amount of melanin produced by the melanocytes. Therefore, as the amount of melanin increases, the brightness decreases, and the first cell group 10 of the skin model 1 exhibits a dark color. Conversely, as the amount of melanin decreases, the brightness increases, and the first cell group 10 of the skin model 1 exhibits a light color. That is, by comparing the brightness of the color of the skin model 1, the therapeutic or preventive effect of the added candidate substance on pigmentation can be evaluated. The brightness can be quantified by recording the first cell group 10 of the skin model 1 as an image and using a known image measurement means.

[0049] In one embodiment, the method of the present invention may further include a step of adding a pigment uptake evaluation substance to the first cell group prior to step (1), and in step (3), the degree of uptake of the pigment uptake evaluation substance into the first cell group may be used as an index of pigmentation, thereby evaluating a factor for treating or preventing skin pigmentation (e.g., Figure 3(A)).

[0050] Moreover, in one embodiment, the method of the present invention may further include a step of adding a pigment uptake evaluation substance to the first cell group after step (2), and in step (3), the degree of uptake of the pigment uptake evaluation substance into the first cell group may be used as an index of pigmentation, thereby evaluating a factor for treating or preventing skin pigmentation (e.g., Figure 2(F)). EXAMPLES

[0051] The present invention will be described in more detail below with reference to examples, but these are not intended to limit the present invention in any way.

[0052] 1. Materials and Experimental Methods 1-1. Epidermal cell culture

[0053] Normal human epidermal melanocytes (Kurabo) were cultured in Medium 254 (Thermo Fisher Scientific) supplemented with human melanocyte growth supplement (HMGS-2).Normal human epidermal keratinocytes (Kurabo) were cultured in Epilife® medium (Thermo Fisher Scientific) containing 60 μM calcium and supplemented with keratinocyte growth supplement (EDGS).

[0054] 1-2. Fibroblast culture and PUVA treatment

[0055] Normal human fibroblasts (Cell Research Corp) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, and 3 × 10 5 Before 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 4 mL of PBS, and the PBS was removed and replaced with 1 mL of psoralen (final concentration 25 ng / mL). In this state, 6 J / cm 2 The cells were irradiated with UVA (hereinafter referred to as "PUVA treatment"). After swelling with 4 mL of PBS, the PBS was removed and replaced with 10% DMEM, and the cells were cultured for 3 days, and the culture supernatant and cells were collected. Fibroblasts derived from the same donor that were not treated with PUVA were used as a control for the PUVA-treated fibroblasts.

[0056] 1-3. Preparation of epidermal cell and fibroblast bilayer culture model

[0057] Keratinocytes or melanocytes were cultured in a 6-well plate at 1x10 5 The cells were seeded and cultured for about 24 hours to allow the cells to adhere (Figure 1(A)). After that, 1 × 10 fibroblasts were seeded per well on the culture insert after 3 days of PUVA treatment or after no treatment. 4The cells were seeded on the culture insert. To induce changes in the epidermal cells that resemble those of pigmented spots, they were co-cultured with PUVA-treated fibroblasts for 3 to 5 days. When examining the difference in the distribution of pigment granules in keratinocytes, fluorescent beads or pigment granules were added at the start of co-culture (Figure 3(A)). After induction of pseudo-pigmented spot epidermal cells, 1 / 10, 1 / 5, 1 / 2, or an equal amount of therapeutic fibroblasts (corresponding to "non-PUVA-FB" in Figure 1(D), Figure 2(D), or Figure 3(D)) were added to the culture insert with respect to the number of PUVA-treated fibroblasts initially seeded, and the cells were cultured for another 3 to 5 days to examine the therapeutic effect of fibroblasts on the pseudo-pigmented spot epidermal cells. When examining phagocytosis in keratinocytes, fluorescent beads (Polysciences Inc., Cat.#19507) or pigment granules (30a) were added simultaneously with the therapeutic fibroblasts (Figure 2(F)). For the culture, a 1:1:1 mixture of 10% DMEM as a fibroblast culture medium, M254 as a melanocyte culture medium, and EpiLife as a keratinocyte culture medium was used.

[0058] 1-4.Immunoantibody staining

[0059] The medium was removed from the 6-well plate of the bilayer culture model, washed with PBS, and then 4% paraformaldehyde (Wako) was added and left to stand at room temperature for about 1 hour to fix the cells. After that, after washing three times with PBS, 1 mL of Immunoblock (registered trademark) (DS Pharma Biomedical Co., Ltd.), a blocking agent for immune experiments, was added per well and left to stand at room temperature for 1 hour. After that, after washing three times with PBS, the primary antibody Ki67 (Abcam) or P63 (Abcam) was diluted 1:100 with diluent (1% BSA-PBS), added to the fixed cells, and left to stand overnight at 4°C. After washing three times with PBS, the secondary antibody Alexa Fluor (registered trademark) 488 (Abcam) was diluted 1:1000, added to the fixed cells, and left to stand at room temperature for 1 hour. After washing three times with PBS again, 500 μl of 0.1% Hoechst (registered trademark) 33342 (Thermo Fisher Scientific Inc.)-PBS solution was added and incubated at 37 ° C. and 5% CO2 for 15 minutes to stain the cell nuclei. Then, the cells were washed three times with PBS, and fluorescence observation and image capture were performed under a microscope. The results of immunofluorescence staining were observed with an LSM700 laser scanning confocal microscope, and images were obtained with Zen2011 software (Carl Zeiss). After four independent experiments, representative photos were shown as results.

[0060] To evaluate the aging index using β-Gal, the Cellular Senescence Detection Kit - SPiDER-βGal (Dojindo Laboratories, Inc.) was used according to the manufacturer's instructions. The medium was removed from the 6-well plate of the bilayer culture model, washed with HBSS, and then 1 ml of Bafilomycin A1 working solution was added and the plate was left to stand in a 5% CO2 incubator at 37°C for 1 hour. Next, 1 ml of SPiDER-βGal working solution was added and the plate was left to stand in a 5% CO2 incubator at 37°C for 30 minutes. The supernatant was removed, washed twice with HBSS, and observed under a fluorescent microscope. After four independent experiments, representative photos are shown as results.

[0061] 1-5. Fontana-Masson staining (FM staining)

[0062] To detect melanin granules, tissue staining was performed using the Fontana-Masson Stain Kit (ScyTek). The Silver Solution included with the Fontana-Masson Stain Kit was diluted 4-fold with ultrapure water, to which a small amount of ammonium hydroxide (Wako) was added, and the solution was heated to 60°C and added to keratinocytes stimulated with fibroblasts and left to stand at room temperature for 45 minutes. The Silver Solution was then washed away with water, and after thorough removal of moisture, the Gold Chloride Solution included with the Kit was added and allowed to react at room temperature for 30 seconds. After washing away the Gold Chloride Solution with water, the Sodium Thiosulfate Solution included with the Kit was added and left to stand at room temperature for 2 minutes. Finally, the Thiosulfate Solution was washed away with water, and the Nuclear Fast Red Solution included with the Kit was added and left to stand at room temperature for 10 minutes to perform nuclear staining. The stained samples were mounted in Mount-Quick and observed under a microscope and photographed. The image analysis software ImageJ (NIH) was used to quantify the area of ​​melanin granules from the photographs.

[0063] 1-6.RT-PCR

[0064] Total RNA was extracted using RNeasy Mini Kit (Qiagen). 200 ng of total RNA was reverse transcribed using PrimeScript RT Reagent Kit (Takara). PCR was performed using PrimeScript RT-PCR Kit (Takara). Gene expression of Ki67, P63, P16, P21 and GAPDH in keratinocytes after fibroblast treatment was analyzed by RT-PCR assay. PCR results were obtained from three independent experiments.

[0065] 1-7. Purification of melanin-rich fraction

[0066] Normal human melanocytes were cultured in a 10 cm dish at 2 × 10 6 The cells were seeded at a concentration of 1000 x g / dish and cultured for 24 hours, after which the medium was collected. The collected medium was centrifuged (room temperature, 2,000 x g, 10 min) to precipitate floating melanocytes and pigment granules, the supernatant was removed, and the medium was resuspended in 1 ml of medium. The suspension was centrifuged again (room temperature, 20,000 x g, 5 min) and the supernatant was removed. The resulting pellet was suspended in 1 ml of medium and added to a hanging cell culture insert (6-well Millicell 8 mm PET) (Millipore, Billerica, MA) attached to a 6 cm dish, and the remaining floating cells were removed through a filter. The collected medium was centrifuged (room temperature, 20,000 x g, 5 min) and used as isolated melanosomes. The pellet was suspended in a small amount of medium and added to the culture medium of TLR-stimulated keratinocytes and cultured for 24 hours, after which melanosome uptake was confirmed by immunofluorescence staining.

[0067] 1-8. Purification of isolated melanosomes

[0068] Confluent melanocytes were harvested and homogenized in homogenization buffer (0.25 M sucrose, 10 mM HEPES-KOH (pH 7.2), 1 mM 2-mercaptoethanol, 1 mM EDTA, EDTA-free protease inhibitor cocktail). The cells were passed 15 times through a 25G needle and centrifuged (4°C, 1000 × g, 10 min). The supernatant was collected and recentrifuged (4°C, 10,000 × g, 10 min), after which the pellet was washed twice with homogenization buffer and centrifuged again (4°C, 10,000 × g, 10 min). The pellet was resuspended in homogenization buffer and used immediately as the melanosome-enriched fraction.

[0069] 1-9.Statistical processing

[0070] Statistical analysis was performed using Graph Pad Prism 5 (Graph Pad Software, La Jolla, CA) by one-way analysis of variance. A statistically significant difference was determined to exist when p<0.05, and the degree of significance is indicated by the following asterisks: (*p<0.05, **p<0.01, ***p<0.001). All experiments were repeated three or more times to confirm reproducibility.

[0071] 2.Results

[0072] 2-1. Study of differentiation and proliferation of epidermal cells To confirm the changes in the differentiation and proliferation levels of keratinocytes, we used keratinocytes co-cultured with cells treated with PUVA Fb or with PUVA Fb plus non-irradiated Fb, and calculated the ratio of cells positive for the differentiation marker p63 and the proliferation marker Ki67 by immunofluorescence staining. As a result, the number of p63- and Ki67-positive cells decreased in the treatment group (Fig. 4), and the expression of each gene was only 10-20% of that in the presence of PUVA Fb alone (Fig. 5). These results indicate that non-irradiated Fb suppresses the abnormal proliferation of keratinocytes and the thickening of the epidermis due to an increase in differentiated cells, which are often observed in spots.

[0073] 2-2. Examination of phagocytic ability of epidermal cells To investigate the effect of co-culture with PUVA-treated fibroblasts (PUVA Fb) and therapeutic fibroblasts (non-irradiated Fb) on the phagocytic activity of keratinocytes, fluorescent beads and melanosomes isolated from melanocytes were added to keratinocytes. Quantification by fluorescence measurement and Fontana-Masson staining confirmed that the uptake of both fluorescent beads and isolated melanosomes into keratinocytes was reduced in a dose-dependent manner by non-irradiated Fb (Figure 6). These results indicate that non-irradiated Fb inhibits the phagocytosis and melanosome-specific uptake that are constitutively performed in keratinocytes.

[0074] 2-3. Examination of changes in melanosome localization in epidermal cells To confirm the localization of melanosomes in keratinocytes, an equal amount of melanosomes was taken up by keratinocytes, and then co-cultured with cells treated with PUVA Fb and non-irradiated Fb in addition to PUVA Fb to confirm the change in localization. As a result, it was found that in the presence of PUVA Fb alone, excess melanosomes were distributed throughout the cytoplasm of keratinocytes, causing uneven pigmentation (Figure 7). In contrast, in the non-irradiated Fb group, melanosomes were distributed uniformly, suppressing the formation of uneven pigmentation.

[0075] To evaluate the senescence level of each keratinocyte, we measured the protein expression of β-galactosidase and the gene expression of senescence markers p16 and p21. As a result, it was found that the addition of non-irradiated Fb reduced the senescence level of keratinocytes (Figures 8 and 9). In addition, excessive accumulation of melanosomes was observed in keratinocytes with high expression of β-galactosidase. [Explanation of symbols]

[0076] 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 30a, 30b pigment granules RD light irradiation FB fibroblasts bFB Fibroblasts damaged by light irradiation KC Keratinocytes MC Melanocyte CA candidate substance

Claims

1. a first cell population comprising keratinocytes and / or melanocytes seeded onto a first cell culture substrate; a second cell group, the second cell group including fibroblasts seeded on a second cell culture substrate having a porous membrane, the second cell group being disposed above the first cell group; A skin model comprising: The fibroblasts are fibroblasts damaged by light irradiation, Soluble components are exchanged between a first culture medium for culturing the first cell group and a second culture medium for culturing the second cell group via the porous membrane. Skin model.

2. The skin model according to claim 1 , wherein the fibroblasts damaged by photoirradiation 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 second cell group further comprises fibroblasts to be evaluated.

6. The skin model according to any one of claims 1 to 5, wherein the first cell group is added with a substance for evaluating pigment uptake.

7. A method for producing a skin model, comprising: A step of disposing a second cell culture substrate having a porous membrane on which a second cell group including fibroblasts damaged by light irradiation is seeded above a first cell culture substrate on which a first cell group including keratinocytes and / or melanocytes is seeded, and co-culturing the first cell group and the second cell group, A step of exchanging soluble components between a first medium for culturing the first cell group and a second medium for culturing the second cell group through the porous membrane. A method comprising:

8. The method according to claim 7 , wherein the fibroblasts damaged by photoirradiation are fibroblasts damaged by irradiation with ultraviolet light in the presence of a photosensitizer.

9. 9. The method of claim 8, 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).

10. The method according to any one of claims 7 to 9, wherein the light irradiation is UVA light irradiation.

11. The method according to any one of claims 7 to 10, wherein the second cell group further comprises fibroblasts to be evaluated.

12. The method according to any one of claims 7 to 11, wherein the first cell group is added with a dye uptake evaluation substance.

13. 1. A method for evaluating an agent for treating or preventing skin pigmentation, comprising: (1) A step of preparing a skin model by disposing a second cell culture substrate having a porous membrane on which a second cell group including fibroblasts damaged by light irradiation is seeded above a first cell culture substrate on which a first cell group including keratinocytes and / or melanocytes is seeded, and co-culturing the first cell group and the second cell group, a step of exchanging soluble components between a first medium for culturing the first cell group and a second medium for culturing the second cell group via the porous membrane; (2) applying a candidate factor to the skin model and culturing it; (3) analyzing an index of pigmentation of the first cell group obtained in the step (2) and evaluating the therapeutic or preventive effect of the candidate factor; A method comprising:

14. The method according to claim 13, further comprising a step of adding a dye uptake evaluation substance to the first cell group prior to step (1), and in step (3), the degree of uptake of the dye uptake evaluation substance into the first cell group is used as an index of the pigmentation.

15. The method according to claim 13, further comprising the step of adding a dye uptake evaluation substance to the first cell group after step (2), and in step (3), the degree of uptake of the dye uptake evaluation substance into the first cell group is used as an index of the pigmentation.

16. The method of any one of claims 13 to 15, wherein the candidate agent is a fibroblast and is applied to the second cell population.

17. The method according to any one of claims 13 to 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 13 to 18, wherein the light irradiation is UVA light irradiation.

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