JP7661133B2 - 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 the skin model.

The skin is an organ that covers the surface of the body 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.

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 (the basal layer) and differentiate into the spinous layer, granular layer, and stratum corneum as they move upwards toward the surface, eventually becoming dandruff and falling off.

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.

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 issues such as not achieving the expected effect, or only a temporary effect being achieved, which is not sustained and leads to recurrence, and there is an ongoing need to develop new treatments or prevention methods and treatments.

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 instead of 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 and has little variation in quality, and that can be evaluated in short- to medium- to long-term experimental systems (e.g., about 3 to 21 days), and a method for producing the same. In addition, the 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.

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 disposed above the first cell group and including fibroblasts seeded on a second cell culture substrate having a porous membrane; and the skin model was found to be stable and has 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).

[1] A first cell group including 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 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 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 includes fibroblasts to be evaluated.
[6] The skin model according to any one of items 1 to 5, wherein a substance for evaluating pigment uptake is added to the first cell group.

[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 method comprising 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.
[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 according to item 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 a fibroblast to be evaluated.
[12] The method according to any one of items 7 to 11, wherein a substance for evaluating dye uptake has been added to the first cell group.

[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 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 Item 13, further comprising a step of adding a dye uptake evaluation substance to the first cell group before the step (1), and in the step (3), a 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), a 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 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 13 to 18, 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 allows confirmation of the formation of pseudo-blemish areas even in short- to medium- to long-term experimental systems (e.g., about 3 to 21 days). In addition, 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.

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. 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. 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 shows the results of investigating the inhibitory effect on keratinocyte proliferation and differentiation when PVUA-untreated fibroblasts were added using the skin model of 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 lower right of each image indicate the percentage of positive cells for each marker. 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 of Figure 1. Gene expression of Ki67 (A) or P63 (B) in keratinocytes after 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 shows the results of investigating the uptake effect 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. Fig. 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 Fig. 3. The state of melanosome uptake in keratinocytes was observed by Fontana-Masson staining. Fig. 8 shows the results of examining keratinocyte aging markers when the fibroblasts to be evaluated (non-PUVA-Fb) were added using the skin model of Fig. 3. Keratinocytes fluorescently stained for β-gal (green) and nuclei (blue) were observed under a fluorescent microscope. Figure 9 shows the results of examining keratinocyte aging markers when the fibroblasts to be evaluated (non-PUVA-Fb) were added using the skin model of Figure 3. Gene expression of p16 (A) or p21 (B) in keratinocytes after 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.

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

In this specification, the terms "first", "second", etc. are used to distinguish one element from another. For example, a first element may be expressed as a second element, and a second element may be expressed as a first element, without departing from the scope of the present invention.

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

<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 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,
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, FIG. 1(C)).

In addition, the skin model 1 in one embodiment 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.

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 differentiate into the spinous layer, granular layer, and stratum corneum as they move upwards, eventually becoming stratum corneum and falling off.

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.

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.

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.

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 composition. The first cell group 10 may contain, for example, only keratinocytes, only melanocytes, or keratinocytes and melanocytes. When both are contained, for example, the ratio of keratinocytes:melanocytes may be 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 to 10 ⁶ cells/cm ² , preferably 1.0 to 10×10 ⁴ cells/cm ² , more preferably about 4 to 8×10 ⁴ cells/cm ² .

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 placed 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. Therefore, 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.

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 any known culture substrate capable of culturing fibroblasts, but is preferably, for example, a cell culture insert.

The average pore size of the porous membrane 22 can be appropriately selected, and may be, for example, an average pore size of about 0.01 μm to about 100 μm (e.g., 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, 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 .

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.

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.

Fibroblasts that are damaged by light irradiation are used as the fibroblasts contained in the second cell group 20. The fibroblasts that are 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, etc.). By using a photosensitizer, the irradiated light is sensitized, and it is possible to efficiently damage 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), 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, it may be irradiated at 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 .

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 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 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.

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.

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.

The skin model 1 can be cultured for 0 to 14 days at approximately 37°C using a culture medium such as KG medium, EpilifeKG2 (Kurabo Industries), Humedia-KG2 (Kurabo Industries), or assay medium (TOYOBO) as the culture medium. 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 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 like the structure of the dermis. In this specification, "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.).

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)).

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 to evaluate 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.

A pigment uptake evaluation substance that can be used in the present invention, such as 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 melanosome-rich fraction. The melanosomes recovered by these methods can be applied to the first cell group to evaluate the uptake action (phagocytosis) of melanosomes.

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 pigment uptake evaluation substances, and melanin production by melanocytes. In addition, 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 as evaluation targets for treating or preventing skin pigmentation.

Methods 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 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;
Contains:

In one embodiment, the candidate factor may be, for example, a low molecular weight 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 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.

In one embodiment, the candidate factor CA may be any cell, such as a fibroblast precursor cell including a mesenchymal stem cell, a fibroblast (e.g., a fibroblast not damaged by light irradiation, or a fibroblast with low damage by light irradiation), as shown in Figures 1(D), 2(D), and 3(D). In this specification, "fibroblasts not damaged by light irradiation" or "fibroblasts with low damage by light irradiation" refers 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 is added to the skin model 1, and after culturing for a desired period of time, the pigmentation index in the skin model 1 is analyzed, thereby enabling the evaluation of the pigmentation treatment or prevention effect of the candidate substance. For example, when 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 treatment or prevention effect is added, the candidate substance can be evaluated as having a pigmentation treatment or prevention effect.

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 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 enhancement of keratinocyte phagocytosis.

In this specification, "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 production 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 production can also 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 a method such as, but not limited to, ELISA, flow cytometer, Western blot, immunohistochemistry, or qPCR.

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. In other words, 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.

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., FIG. 3(A)).

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., FIG. 2(F)).

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.

1. Materials and experimental methods used 1-1. Cultivation of epidermal cells

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).

1-2. Fibroblast cell culture and PUVA treatment

Normal human fibroblasts (Cell Research Corp) were cultured in 10% fetal bovine serum-containing Dulbecco's Modified Eagle Medium (hereinafter, 10% DMEM) and 3 x 10 ⁵ cells were seeded in a T25 flask. Before the cell density reached 100% confluence, the medium was replaced with 10% DMEM containing 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 PBS containing psoralen (final concentration 25 ng/mL), and 6 J/cm ² UVA irradiation was performed (hereinafter, "PUVA treatment"). After that, the cells were swollen 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 but not treated with PUVA were used as a control for the PUVA-treated fibroblasts.

1-3. Creation of a bilayer culture model of epidermal cells and fibroblasts

Keratinocytes or melanocytes were seeded at ^1x105 cells each onto a 6-well plate and cultured for about 24 hours to allow the cells to adhere (Fig. 1(A)). Then, ^1x104 cells per well of fibroblasts either 3 days after PUVA treatment or untreated were seeded onto the culture insert. To induce changes in epidermal cells resembling spots, the cells were co-cultured with PUVA-treated fibroblasts for 3-5 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 (Fig. 3(A)). After induction of pseudo-age 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 in an amount of 1/10, 1/5, 1/2, or the same amount as the number of PUVA-treated fibroblasts initially seeded, and cultured for another 3 to 5 days to examine the therapeutic effect of fibroblasts on pseudo-age 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, a fibroblast culture medium, M254, a melanocyte culture medium, and EpiLife, a keratinocyte culture medium, was used.

1-4. immunoantibody staining

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 allowed to stand at room temperature for about 1 hour to fix the cells. After that, after washing three times with PBS, 1 mL of an immune experiment blocking agent, Immunoblock (registered trademark) (DS Pharma Biomedical Co., Ltd.) was added per well and allowed 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 allowed to stand overnight at 4°C. After washing three times with PBS, the secondary antibody Alexa Fluor (registered trademark) 488 (Abcam) was diluted at a ratio of 1:1000, added to the fixed cells, and allowed to stand at room temperature for 1 hour. After washing again three times with PBS, 500 μl of 0.1% Hoechst (registered trademark) 33342 (Thermo Fisher Scientific Inc.)-PBS solution was added and incubated for 15 minutes under 37 ° C. and 5% CO ₂ conditions 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.

To evaluate the aging index by β-Gal, Cellular Senescence Detection Kit-SPiDER-βGal (Dojindo Laboratories, Inc.) was used according to the manufacturer's instructions. After removing the medium from the 6-well plate of the bilayer culture model and washing with HBSS, 1 ml of Bafilomycin A1 working solution was added and left to stand for 1 hour in a 5% CO ₂ incubator at 37 ° C. Then, 1 ml of SPiDER-βGal working solution was added and left to stand for 30 minutes in _a 5% CO 2 incubator at 37 ° C. The supernatant was removed, washed twice with HBSS, and observed under a fluorescent microscope. After four independent experiments, representative photographs were shown as results.

1-5. Fontana Masson staining (FM staining)

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 then heated to 60°C and added to keratinocytes stimulated with fibroblasts, and allowed 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. Furthermore, after rinsing off the Gold Chloride Solution with water, the Sodium Thiosulfate Solution included in the kit was added and left to stand at room temperature for 2 minutes. Finally, the Thiosulfate Solution was washed off with water, and the Nuclear Fast Red Solution included in the kit was added and left to stand at room temperature for 10 minutes to perform nuclear staining. After staining, the samples were mounted in Mount-Quick and observed and photographed under a microscope. The image analysis software ImageJ (NIH) was used to quantify the area of melanin granules from the photographs.

1-6. RT-PCR

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.

1-7. Purification of melanin-rich fraction

Normal human melanocytes were seeded in a 10 cm dish at a concentration of 2 x 10 ⁶ cells/dish, and the medium was collected after 24 hours of culture. The collected medium was centrifuged (room temperature, 2,000 x g, 10 minutes) to precipitate floating melanocytes and pigment granules, and the supernatant was removed and then resuspended in 1 ml of medium. The suspension was centrifuged again (room temperature, 20,000 x g, 5 minutes) 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 minutes) and used as isolated melanosomes. The pellet was suspended in a small amount of medium and added to a culture medium for TLR-stimulated keratinocytes. After culturing for 24 hours, melanosome uptake was confirmed by immunofluorescence staining.

1-8. Purification of isolated melanosomes

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). They 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 precipitate 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.

1-9. Statistical processing

Statistical analysis was performed using Graph Pad Prism 5 (Graph Pad Software, La Jolla, CA) with one-way analysis of variance. A statistically significant difference was determined when p<0.05, and the degree of statistical 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. Results

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

2-2. Study of phagocytic activity of epidermal cells To study 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 phagocytosis and melanosome-specific uptake, which are constantly performed in keratinocytes.

2-3. Study of changes in melanosome localization in epidermal cells In order to confirm the localization of melanosomes in keratinocytes, an equal amount of melanosomes was taken up by keratinocytes, and then co-cultured with PUVA Fb and a cell group to which non-irradiated Fb was added to PUVA Fb, and changes in localization were confirmed. As a result, it was found that in the presence of only PUVA Fb, keratinocytes in which excess melanosomes were diffused throughout the cytoplasm were partially localized, causing uneven coloring (Figure 7). In contrast, in the group to which non-irradiated Fb was added, melanosomes were distributed uniformly, and the formation of unevenness was suppressed.

To evaluate the aging level of each keratinocyte, the protein expression of β-galactosidase and the gene expression of aging markers p16 and p21 were measured, and it was found that the addition of non-irradiated Fb reduced the aging level of keratinocytes (Figures 8 and 9). In addition, excessive accumulation of melanosomes was observed in keratinocytes with high expression levels of β-galactosidase.

Reference Signs List 1, 1a, 1b Skin model 10 First cell group 11 First cell culture substrate 12 First culture medium 20 Second cell group 21 Second cell culture substrate 22 Porous membrane 23 Second culture medium 30a, 30b Pigment granules RD Light irradiation FB Fibroblasts bFB Fibroblasts damaged by light irradiation KC Keratinocytes MC Melanocytes CA Candidate substance

Claims (19)

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. 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. 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 second cell group further includes fibroblasts to be evaluated. 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. 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 method comprising 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. The method according to claim 7, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer. 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). The method according to any one of claims 7 to 9, wherein the light irradiation is UVA light irradiation. The method according to any one of claims 7 to 10, wherein the second cell group further includes fibroblasts to be evaluated. The method according to any one of claims 7 to 11, wherein the first cell group is added with a dye uptake evaluation substance. 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: The method according to claim 13, further comprising a step of adding a dye uptake evaluation substance to the first cell group before 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. The method according to claim 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. The method according to any one of claims 13 to 15, wherein the candidate factor is a fibroblast and is applied to the second cell group. 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. 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 13 to 18, wherein the light irradiation is UVA light irradiation.

Images