JP7733479B2 - Skin model and method for producing same, and method for evaluating factors for treating or preventing skin pigmentation - Patents.com - Google Patents

Description

The present invention relates to a skin model and a method for producing the same. The present invention also relates to a method for evaluating factors for treating or preventing skin pigmentation using a skin model.

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 harmful substances from entering the body, and plays an essential role in maintaining life.

The skin of higher vertebrates is broadly divided into the epidermis, dermis, and subcutaneous tissue layers, starting from the outermost layer. The epidermis is primarily composed of cells called keratinocytes, which divide in the deepest part of the epidermis (the basal layer) and differentiate into the spinous layer, granular layer, and stratum corneum as they move upward, eventually becoming stratum corneum and being shed.

Melanocytes (pigment cells) reside 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. As keratinocytes turnover, the taken-up melanin migrates to the stratum corneum and is excreted from the body over a period of approximately 40 days.

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 result in the excessive production of melanin 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 and resulting in recurrence. Therefore, there is a continuing need for the development of new treatments, prevention methods, and therapeutic agents.

In light of this situation, 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 as an alternative to conventional animal models (e.g., Patent Documents 1 and 2).

Patent No. 6113393 International Publication No. 2020/111265

The 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., approximately 3 to 21 days), as well as a method for producing the same. The object of the present invention is also to provide a method for evaluating candidate factors for treating or preventing skin pigmentation using the new skin model.

As a result of extensive research by the present inventors, it has been found that a first cell group comprising fibroblasts seeded on a cell culture substrate;
It has been found that a skin model comprising: a soluble component-permeable porous membrane having a second cell group including melanocytes seeded on one side thereof and a third cell group including keratinocytes seeded on the other side thereof, the porous membrane being positioned 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.

[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 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, 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 light irradiation 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 includes fibroblasts to be evaluated.

[8] A method for producing a skin model, comprising:
A method comprising the steps of: placing 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.
[9] The method according to Item 8, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.
[10] The method according to 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.

[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) A step of 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] 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 light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer.
[18] The method according to 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 a porous membrane 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.

The present invention provides a skin model that is stable and has little variation in quality, and that makes it possible to confirm the formation of pseudo-blemish areas even in short- to medium- to long-term experimental systems (e.g., approximately 3 to 21 days). Furthermore, the use of the skin model provided by the present invention makes it possible to search for and evaluate candidate factors for treating or preventing skin pigmentation.

1 is a schematic diagram showing a method for manufacturing a skin model according to one embodiment, with each component primarily shown in cross section. Figure 2 shows the results of measuring the intracellular melanin content using the skin model of Figure 1. Top: photographs of the appearance of equal amounts of melanocyte lysates, bottom: results of measuring equal amounts of melanocyte lysates at OD 405 nm. Figure 3 shows the relative expression levels (the level in the case of PUVA-Fb alone is 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 of Figure 1. *: p<0.05, **: p<0.01, ***: p<0.01. Figure 4 shows the relative expression levels (the level in the case of PUVA-Fb alone is 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 of Figure 1. *: p<0.05, **: p<0.01.

The following describes in detail the embodiments of the present invention with reference to the drawings, etc., but the technical scope of the present invention is not limited to the embodiments described below.

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 referred to as a second element, and similarly, a second element may be referred to as a first element, without departing from the scope of the present invention.

Unless otherwise defined, terms (technical and scientific) used herein have the same meaning as commonly understood by those skilled in the art.

<Skin model and method for producing same>
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 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,
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)).

In addition, the skin model 1 in one embodiment is
The present invention can be provided by a method including the steps of: placing 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.

Keratinocytes are one of the cells that make up the epidermis. In the epidermal tissue of a living body, they divide in the deepest layer (the basal layer) and differentiate upward into the spinous layer, granular layer, and then stratum corneum, before migrating to the surface and eventually becoming sebum and being shed.

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

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

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.

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.

The fibroblasts contained in the first cell group 10 are fibroblasts that have been damaged by light irradiation. 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.

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), 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 adjusted appropriately depending on the wavelength, irradiation time, cell density, etc. For example, when irradiating with UVA, the intensity of the light may be 0.01 J/cm ² to 100 J/cm ² , preferably 0.1 J/cm ² to 20 J/cm ² , and more preferably 0.5 J/cm ² to 10 J/cm ² .

Culturing light-irradiated fibroblasts for a certain period of time prevents the induction of apoptosis and allows only surviving fibroblasts to proliferate (corresponding to (A) in Figure 1). Fibroblasts damaged by light irradiation exhibit 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 assessed by measuring commonly known cellular senescence markers, such as the expression level of senescence-associated acid β-galactosidase (SA-βgal), the constitutive activation of cell cycle control 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 the desired level of senescence can be obtained by adjusting the amount of light irradiation.

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

In one embodiment, a second cell group 20 comprising melanocytes that constitutes the skin model 1 is seeded on one side of a porous membrane 22 using a second cell culture substrate 21 having a porous membrane 22 that is permeable to soluble components (e.g., FIG. 1(C)). After the second cell group 20 adheres 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.

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, such as ad-MED Vitrigel from Kanto Chemical Co., Ltd. (Japan) or the permeable collagen membrane from Koken Co., Ltd. (Japan).

The average pore size of the porous membrane 22 may be, for example, about 0.01 μm to about 100 μm (eg, 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 selected appropriately, and may be, for example, 1×10 ⁴ /cm ² or more, 1×10 ⁵ /cm ² or more, 5× ^{10 5 /} cm ² or ^more , 10×10 ⁵ /cm ² or more, 50×10 ⁵ /cm ² or more, or 100×10 ⁵ /cm ² or more, or 1×10 4 /cm 2 to 100×10 ⁸ /cm ² , or 1×10 ⁵ /cm ² to 100×10 ⁸ /cm ² .

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 contained in the epidermis. The number of cells seeded in the skin model 1 is not particularly limited, and may be, for example, 1 x ¹⁰ to ¹⁰ cells/ ^cm , 1.0 to 10 x ¹⁰ cells/ ^cm , or approximately 4 to 8 x ¹⁰ cells/ ^cm .

In the skin model 1 of the present invention, a second cell culture substrate 21, on 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 the first cell group 10. The first cell group 10, second cell group 20, and third cell group 30 are co-cultured in the same medium without being mixed with each other. This allows for the exchange of soluble components produced by the first cell group 10, second cell group 20, and third cell group 30, respectively.

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).

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), Humedia-KG2 (Kurabo Industries), or assay medium (TOYOBO). Other culture media that can be used include DMEM medium (GIBCO) or a 1:1 mixture of ascorbic acid-containing KGM and DMEM.

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.).

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, leading to hyperproliferation, hyperdifferentiation, or uptake of pigment granules by keratinocytes, 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.

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

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) A step of 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;
Includes:

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., a herbal extract, a compound derived from a herbal drug), and a microbial compound, extract, or culture product.

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 with low damage by light irradiation), as shown in Figure 1(F). As used herein, "fibroblasts not damaged by light irradiation" or "fibroblasts with low damage 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."

The candidate substance's therapeutic or preventive effect on pigmentation can be evaluated by adding the candidate substance to the skin model 1 and analyzing the pigmentation index in the skin model 1 after culturing for a desired period of time. For example, if the pigmentation index improves in the skin model 1 to which the candidate substance has been added, compared with the pigment production and/or pigmentation level in the skin model 1 to which the candidate substance has not been added or to which any substance that does not have a therapeutic or preventive effect on pigmentation has been added, the candidate substance can be evaluated as having a therapeutic or preventive effect on pigmentation.

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.

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. Furthermore, the amount of pigment produced can be measured, for example, by measuring the amount of melanin or the nucleic acid encoding it (e.g., mRNA amount) 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.

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 changes 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 known image measurement means.

The present invention will be explained in more detail below based on examples, but these examples are not intended to limit the present invention in any way.

1. Materials and Experimental Methods Used

1-1. Epidermal cell culture

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).

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 seeded at 1 x ¹⁰ cells per well in a 6-well plate. Before the cell density reached 100% confluence, the medium was 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 irradiated with 6 J/cm ² of UVA (hereinafter referred to as PUA treatment). The cells were then swollen with 1 mL of PBS, the PBS was removed, and the medium was replaced with 10% DMEM. After culturing for 3 days, the culture supernatant and cells were collected. PUVA-untreated fibroblasts from the same donor served as a control for the PUVA-treated fibroblasts.

1-3. Creation of a non-contact trilayer culture model of keratinocytes, pigment cells, and fibroblasts

To create the triple-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 seeded on one side of the collagen membrane at 1 x ¹⁰⁴ to 1 x ¹⁰⁵ cells and cultured for 24 hours to allow them to adhere to the membrane. Keratinocytes were then seeded on the other side at 1 x ¹⁰⁴ to 1 x ¹⁰⁵ cells. PUVA-treated fibroblasts were previously seeded at 1 x ¹⁰⁴ cells/well in a 24-well plate. The collagen membrane with the attached keratinocytes and melanocytes was then placed on top of the fibroblasts and co-cultured for 3 to 5 days to induce changes in the epidermal cells resembling pigmented spots. After induction of pseudo-age spot epidermal cells, therapeutic fibroblasts (corresponding to "non-PUVA-FB" in Figure 1(F)) were added to a 24-well plate at 1/10, 1/5, 1/2, and an equal amount of the PUVA-treated fibroblasts initially seeded, and cultured for an additional 3 to 5 days to examine the therapeutic effect of fibroblasts on pseudo-age spot epidermal cells. For culture, a 1:1:1 mixture of 10% DMEM (fibroblast culture medium), M254 (melanocyte culture medium), and EpiLife (keratinocyte culture medium) was used.

1-4. Measurement of intracellular and extracellular melanin content

Melanocytes were seeded at 1 x 105 ^cells /well on one side of the collagen gel and cultured using the model described above. The medium was collected and centrifuged (4°C, 200 x g, 10 minutes) to remove cell debris. The resulting supernatant was measured at OD405 nm to determine the amount of extracellular melanin. Melanocytes were also collected by trypsinization and centrifuged (4°C, 2,300 x g, 5 minutes). 120 μl of 0.2 N NaOH was added to the resulting cell mass, and the mixture was heated at 80°C for 30 minutes to extract intracellular melanin. The resulting supernatant was measured at OD405 nm to determine the amount of intracellular melanin. Results were obtained from three independent experiments (Figure 2).

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 was analyzed by RT-PCR assay. PCR results were obtained from three independent experiments (Figures 3 and 4). In this experiment, the analysis was performed using the following primer sequences:

1-6. Statistical Processing

Statistical analysis was performed using Graph Pad Prism 5 (Graph Pad Software, La Jolla, CA) with one-way analysis of variance. A p < 0.05 was considered to indicate statistical significance, and the level 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.

2. result

Melanocytes and keratinocytes were collected from the skin model, and the intracellular melanin content and gene expression involved in melanin synthesis were measured. It was found that the increase in melanin content was suppressed in the model with non-irradiated Fb compared to the model with PUVA Fb alone. RT-PCR analysis demonstrated that this suppression of darkening was due to a decrease in the gene expression of the melanin synthesis enzyme tyrosinase, tyrosinase-related genes TYRP1 and DCT, and their transcription factor MITF, which are expressed in melanocytes. Furthermore, in keratinocytes, the gene expression of SCF and ET1, which induce melanin synthesis, decreased in a concentration-dependent manner with non-irradiated Fb. These results demonstrate that non-irradiated Fb acts on both melanocytes and keratinocytes to suppress melanin synthesis induced by PUVA Fb.

1, 1a, 1b Skin model 10 First cell population 11 First cell culture substrate 12 First culture medium 20 Second cell population 21 Second cell culture substrate 22 Porous membrane 23 Second culture medium 24 Third cell culture substrate 30 Third cell population RD Light irradiation FB Fibroblast bFB Fibroblast damaged by light irradiation KC Keratinocyte MC Melanocyte CA Candidate substance

Claims (21)

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 . The skin model of claim 1, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer. 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). The skin model according to any one of claims 1 to 3, wherein the light irradiation is UVA light irradiation. The skin model according to any one of claims 1 to 4, wherein the porous membrane is made of a biocompatible component. The skin model according to any one of claims 1 to 5, wherein the porous membrane is a collagen membrane. The skin model described in any one of claims 1 to 6, wherein the first cell group further includes fibroblasts to be evaluated. A method for producing a skin model for confirming pseudo-blemish sites formed by fibroblasts damaged by light irradiation , comprising:
A method comprising the steps of: placing 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. The method of claim 8, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer. 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). The method according to any one of claims 8 to 10, wherein the light irradiation is UVA light irradiation. The method according to any one of claims 8 to 11, wherein the porous membrane is made of a biocompatible component. The method according to any one of claims 8 to 12, wherein the porous membrane is a collagen membrane. The method of any one of claims 8 to 13, wherein the first cell group further includes fibroblasts to be evaluated. 1. A method for evaluating a factor for treating or preventing skin pigmentation, comprising:
(1) A step of preparing a skin model for confirming the pseudo-spot 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) A step of 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: The method of claim 15, wherein the candidate factor is a fibroblast and is applied to the first cell group. The method of 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. 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). The method according to any one of claims 15 to 18, wherein the porous membrane is made of a biocompatible component. The method according to any one of claims 15 to 19, wherein the porous membrane is a collagen membrane. The method according to any one of claims 15 to 20, wherein the light irradiation is UVA light irradiation.