JP2022184021A - Skin model and method for producing the same, and method for evaluating factor for treating or preventing skin pigmentation - Google Patents

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

SKIN MODEL AND METHOD FOR MANUFACTURING THE SAME The present invention relates to a skin model and a method for producing the same. The present invention also relates to methods for evaluating factors for treating or preventing skin pigmentation using skin models.

The skin is an organ covering the body surface that separates the in-vivo and in-vitro environments. The skin acts as a physical barrier to protect against dryness and entry of harmful substances into the body, and plays an essential role in sustaining life.

The skin of higher vertebrates is composed of layers of epidermis, dermis, and subcutaneous tissue, roughly divided from the outermost layer. The epidermis is mainly composed of cells called keratinocytes (keratinocytes), and while keratinocytes divide in the deepest part of the epidermis (basal layer), they divide into the stratum spinosum, stratum granulosum, and stratum corneum in the upper layers. As it differentiates, it moves to the surface and eventually falls off as dirt.

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

Pigmentation such as spots and freckles on the skin is caused by the excessive formation of melanin in melanocytes and the deposition of melanin granules in keratinocytes in the basal layer of the epidermis due to hormonal abnormalities, UV exposure, local inflammation, etc. is thought to be the cause. Although methods and therapeutic agents (whitening agents) for treating or preventing pigmentation, such as senile pigment spots due to aging, have been developed, the expected effects are not obtained or the effects are temporary. However, there are problems such as recurrence due to lack of long-lasting effects, and there continues to be a need to develop new treatment or preventive methods and therapeutic agents.

Under such circumstances, skin models that mimic the structure and functions of the skin have been developed and used in recent years instead of conventional animal models to develop new therapeutic methods (e.g., , Patent Documents 1 and 2).

Japanese Patent No. 6113393 WO2020/111265

An object of the present invention is to provide a new skin model that is stable, has little variation in quality, and can be evaluated even in a short- to medium-term (for example, about 3 to 21 days) experimental system, and a method for producing the same. That is. It is also an object of the present invention to provide methods of evaluating candidate agents for treating or preventing skin pigmentation using new skin models.

As a result of extensive studies by the present inventors, a first cell group containing keratinocytes and / or melanocytes seeded on the first cell culture substrate;
A second cell group containing fibroblasts arranged above the first cell group and seeded on a second cell culture substrate having a porous membrane; It has been found that it can be a skin model that can be evaluated even in a short- to medium- and long-term (for example, about 3 to 21 days) experimental system. That is, the present invention includes the following inventions.

[1] a first cell group comprising keratinocytes and/or melanocytes seeded on a first cell culture substrate;
a second cell group containing fibroblasts arranged above the first cell group and seeded on a second cell culture substrate having a porous membrane;
A skin model comprising
The fibroblasts are fibroblasts that have been 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 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 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 group-protected gold nanoclusters (AUxSRy). model.
[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 contains fibroblasts to be evaluated.
[6] The skin model according to any one of items 1 to 5, wherein the first cell group is added with a substance for evaluation of dye uptake.

[7] A method for producing a skin model, comprising:
Above the first cell culture substratum seeded with the first cell group containing keratinocytes and/or melanocytes, the porous membrane is seeded with the second cell group containing fibroblasts damaged by light irradiation. A step of placing a second cell culture substrate and co-cultivating the first cell group and the second cell group,
wherein soluble components are exchanged between a first medium for culturing the first population of cells and a second medium for culturing the second population of cells 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 group-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 contains fibroblasts to be evaluated.
[12] The method according to any one of Items 7 to 11, wherein the first cell group is added with a dye uptake evaluation substance.

[13] A method of evaluating a factor for treating or preventing skin pigmentation, comprising:
(1) Above the first cell culture substrate seeded with the first cell group containing keratinocytes and / or melanocytes, the second cell group containing fibroblasts damaged by light irradiation is seeded Porous A step of placing a second cell culture substrate having a membrane and co-cultivating the first cell group and the second cell group to prepare a skin model,
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;
(3) analyzing the pigmentation index of the first cell group obtained in the step (2) to evaluate the therapeutic or preventive effect of the candidate factor;
A method, including
[14] The step of adding a dye uptake evaluation substance to the first cell group before the step (1), and adding the dye uptake evaluation substance to the first cell group in the step (3). 14. A method according to item 13, wherein the degree of incorporation into the pigmentation is used as an indicator of the pigmentation.
[15] further comprising the step of adding a dye uptake evaluation substance to the first cell group after the step (2), and adding the dye uptake evaluation substance to the first cell group in the step (3); 14. A method according to item 13, wherein the degree of uptake is used as an indicator 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] 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. The method described in section.
[18] The method of item 17, wherein the photosensitizer is selected from the group consisting of psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, and thiol group-protected gold nanoclusters (AUxSRy). .
[19] The method according to any one of items 13 to 18, wherein the light irradiation is UVA light irradiation.

INDUSTRIAL APPLICABILITY 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-blemishes even in short- to medium- and long-term (for example, about 3 to 21 days) experimental systems. be. 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.

FIG. 1 is a schematic diagram showing a method for manufacturing a skin model according to one embodiment. Each component mainly represents a cross section. FIG. 2 is a schematic diagram showing a method of manufacturing a skin model according to one embodiment. Each component mainly represents a cross section. FIG. 4 is a schematic diagram showing a method of manufacturing a skin model according to one embodiment. Each component mainly represents a cross section. FIG. 4 shows the results of examining the keratinocyte proliferation/differentiation inhibitory effect when PVUA-untreated fibroblasts were added using the skin model of FIG. (A) Fluorescence microscope image of keratinocytes fluorescently stained with proliferation markers Ki67 (green) and nuclei (blue). (B) Fluorescent microscope image of keratinocytes fluorescently stained with p63 (green) and nuclei (blue), which are differentiation markers. The numbers on the bottom right of each image indicate the percentage of positive cells for each marker. FIG. 5 shows the results of examining the keratinocyte proliferation/differentiation inhibitory effect when PVUA-untreated fibroblasts were added using the skin model of FIG. 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 and normalized as an internal standard. Each graph represents a relative value when the expression level of each gene in PVUA-untreated fibroblasts and keratinocytes not co-cultured (corresponding to "PVUA") is set to 1. *: p<0.05, **: p<0.01. FIG. 6 shows the results of examining the effect of 0.5 μm beads or melanosome uptake by keratinocytes when fibroblasts (non-PUVA-Fb) to be evaluated were added using the skin model of FIG. (A) Fluorescence microscopy images showing the location 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 fibroblasts (non-PUVA-Fb) to be evaluated were added using the skin model of FIG. The state of melanosome uptake in keratinocytes was observed by Fontana Masson staining. FIG. 8 shows the results of examining keratinocyte aging markers when fibroblasts (non-PUVA-Fb) to be evaluated were added using the skin model of FIG. Keratinocytes with fluorescently stained β-gal (green) and nuclei (blue) were observed under a fluorescence microscope. FIG. 9 shows the results of examining keratinocyte aging markers when fibroblasts (non-PUVA-Fb) to be evaluated were added using the skin model of FIG. 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 and normalized as an internal standard. Each graph represents a relative value when the expression level of each gene in PVUA-untreated fibroblasts and keratinocytes not co-cultured (corresponding to "PVUA") is set to 1. *: p<0.05, **: p<0.01.

EMBODIMENT OF THE INVENTION Hereinafter, although the form for implementing this invention is demonstrated in detail, referring drawings etc., the technical scope of this invention is not limited only to the following forms.

In this specification, terms such as “first” and “second” are used to distinguish one element from another, for example, the first element is expressed as the second element, Similarly, a second element could be termed a first element without departing from the scope of the invention.

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

<Skin model and its manufacturing method>
FIG. 1 is a schematic diagram illustrating a skin model 1 and a method of manufacturing it in one embodiment. In one embodiment, the skin model 1 is
a first cell group comprising keratinocytes and/or melanocytes seeded on the first cell culture substrate;
a second cell group containing fibroblasts arranged above the first cell group and seeded on a second cell culture substrate having a porous membrane;
a skin model,
The fibroblasts are fibroblasts that have been damaged by light irradiation,
A skin model 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 via the porous membrane (e.g., FIG. 1(C)).

In addition, the skin model 1 in one embodiment is
Above the first cell culture substratum seeded with the first cell group containing keratinocytes and/or melanocytes, the porous membrane is seeded with the second cell group containing fibroblasts damaged by light irradiation. A step of placing a second cell culture substrate and co-cultivating the first cell group and the second cell group,
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; can be done.

Keratinocytes (keratinocytes) are one of the cells that make up the epidermis. These cells migrate to the surface while differentiating into layers, and eventually become dirt and fall off.

Melanocytes (pigment cells) are one of the cells that constitute the epidermal tissue, and are cells that exist in the basal layer of the epidermis in vivo and form melanin.

Fibroblasts, keratinocytes and melanocytes used in the present invention may be primary cultured cells collected from living tissue, or may be pre-isolated and/or proliferated commercially available or distributed cells. They may be established cells, or 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 culture substrate 11 is seeded with a first cell group 10 containing keratinocytes and/or melanocytes. The first cell culture substrate 11 is not particularly limited as long as it is a known culture substrate.

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 contained in the epidermal composition Cells and Merkel cells may be included. The first cell group 10 may contain, for example, only keratinocytes, only melanocytes, or both keratinocytes and melanocytes. When both are included, for example, keratinocyte:melanocyte cells may be included in a ratio of 1:1 to 1000:1, preferably 3:1 to 30:1. The number of seeded cells in the first cell group 10 is not particularly limited, but is, 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 10 cells/cm 2 . It may contain 8×10 ⁴ pieces/cm ² .

In the skin model 1 of the present invention, a second cell group 20 containing fibroblasts seeded on a second cell culture substrate 21 having a porous membrane 22 is arranged above the 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 the first medium 12 for culturing the first cell group 10 and the second 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 the porous membrane 22 on which the second cell group 20 is seeded may be a known culture substrate capable of culturing fibroblasts. , preferably a cell culture insert.

The average pore size of the porous membrane 22 can be selected as appropriate, and is, for example, an average pore size of 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, 0.01 μm to 10 μm, 0.01 μm to 100 μm, 0.01 μm to 10 μm, 0.01 μm to 100 μm, 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 selected as appropriate ^. The pore density may be 10 ⁵ /cm ² or more, 50×10 ⁵ /cm ² or more, or 100×10 ⁵ /cm ² or more, and may be 1×10 ⁴ /cm ² to 100×10 ⁸ /cm 2 . ² , 1×10 ⁵ /cm ² to 100×10 ⁸ /cm ² .

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 constitute connective tissue and are present in many organs and tissues. Fibroblasts are mainly contained in the dermis tissue in the skin. The fibroblasts used in the skin model 1 of the present invention are preferably dermis-derived fibroblasts.

Fibroblasts that have been damaged by light irradiation are used as the fibroblasts contained in the second cell group 20 . Fibroblasts damaged by photoirradiation are preferably photoirradiated and damaged in the presence of a photosensitizer. Examples of photosensitizers that can be used include psoralen, NAD, riboflavin, tryptophan, folic acid, porphyrin, methylene blue, thiol group-protected gold nanoclusters (AUxSRy, etc.). The irradiated light is sensitized and can efficiently damage fibroblasts.

The light used to damage fibroblasts damages intracellular nucleic acids, such as DNA and RNA, but may have a wavelength that does not kill all cells. ), more preferably UVA (about 320 nm to about 400 nm). The intensity of the light to be irradiated may damage intracellular nucleic acids such as DNA and RNA, but may be such that apoptosis or the like is induced and all cells are not killed. Wavelength, irradiation time, cell density, etc. can be adjusted as appropriate. For example, when UVA is irradiated, 0.01 J/cm ² to 100 J/cm ² , preferably 0.1 J/cm ² to 20 J/cm ² , more preferably 0.5 J/cm ² to 10 J/cm ² . do it.

By culturing the light-irradiated fibroblasts for a certain period of time, apoptosis or the like is not induced, and only surviving fibroblasts can be grown (corresponding to (B) in FIG. 1). Fibroblasts damaged by light irradiation undergo cellular senescence, e.g., elongated cell morphology, decreased proliferative capacity, and increased production of melanogenic factors (such as stem cell growth factor (SCF)). Shows features with increasing levels. The level of cell senescence is determined by commonly known cell senescence markers, for example, the expression level of senescence-associated acid β-galactosidase (SA-βgal), constant activation of cell cycle check mechanisms such as p21/p53 pathway and p16 pathway, IL It can be examined by measuring the expression level of senescence-associated secretory phenotype (SASP) factors such as -6. A desired level of senescence of fibroblasts can be obtained by adjusting the amount of light irradiation.

Fibroblasts contained in the second cell group 20 that constitutes 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 substantially homogeneous fibroblasts, and can provide a stable system with little variation in activity.

In one embodiment, the cells contained in the first cell group 10 include, for example, cells contained in commercially available TESTSKIN (trademark) LSE-melano (TOYOBO), MelanoDerm (trademark) (MatTek), etc. Keratinocytes may also be used.

For the skin model 1, for example, a culture medium used for normal keratinocyte culture as a culture medium, such as KG medium, Epilife KG2 (Kurabo), Humedia-KG2 (Kurabo), assay medium (TOYOBO), etc., is used at about 37 ° C. from 0 to It can be done over 14 days. As a medium, a DMEM medium (GIBCO) or a 1:1 mixed medium of ascorbic acid-containing KGM and DMEM can be used.

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

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 (for example, 30a in FIG. 2 (F), or 30b) in FIG. 3(A). The pigment uptake evaluation substance may be added after seeding the first cell group 10 containing keratinocytes and/or melanocytes on the first cell culture substrate 11 (for example, FIG. 3(A)), After the skin model 1 is produced, it may be added later to the first cell culture substrate 11 seeded with the first cell group 10 (eg, FIG. 2(F)).

Pigment uptake evaluation substances that can be applied in the present invention include, for example, beads and pigment granules such as melanosomes. The bead material is not particularly limited as long as it can be used for evaluation of uptake of pigment granules. For example, commercially available fluorescent beads made of polystyrene or latex can be used. Also, the size of the beads may be any size that mimics pigment granules, such as melanosomes, for example, 0.01 μm to 10 μm, preferably 0.1 μm to 5 μm, more preferably 0.5 μm to 2 μm (eg, about 1 μm). can be used. This makes it possible to evaluate bead uptake (phagocytosis) by the first cell group.

Pigment uptake evaluation substances applicable 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 may be prepared by homogenizing melanocytes and collecting a melanosome-rich fraction. Melanosomes collected by these methods can be applied to the first cell group to evaluate melanosome uptake (phagocytosis).

In the skin model 1 provided by the present invention, fibroblasts damaged by light irradiation act directly or indirectly, and factors that form skin pigmentation, such as keratinocyte hyperproliferation, hyperdifferentiation, Alternatively, various factors such as the uptake of a pigment uptake evaluation substance and the production of melanin by melanocytes can be evaluated. In addition, by measuring and analyzing the "pigmentation index" in the skin model 1, it is possible to evaluate the factors affecting pigmentation.

By using the skin model 1 of the present invention, for example, it becomes possible to evaluate a candidate factor as an evaluation target for treating or preventing skin pigmentation.

<Method for evaluating factors for treating or preventing skin pigmentation>
The present invention can provide methods for evaluating factors for treating or preventing skin pigmentation using skin models. In one embodiment, the method of the invention comprises:
(1) Above the first cell culture substrate seeded with the first cell group containing keratinocytes and / or melanocytes, the second cell group containing fibroblasts damaged by light irradiation is seeded Porous A step of placing a second cell culture substrate having a membrane and co-cultivating the first cell group and the second cell group to prepare a skin model,
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;
(3) analyzing the pigmentation index of the first cell group obtained in the step (2) to evaluate the therapeutic or preventive effect of the candidate factor;
contains.

In one embodiment, the candidate agent is, for example, a small molecule compound, peptide, nucleic acid, protein, mammalian (eg, mouse, rat, porcine, bovine, ovine, monkey, human, etc.) cell, tissue extract, or cell culture. It may be a supernatant, plant-derived compounds or extracts (eg, crude drug extracts, crude drug-derived compounds), and microorganism-derived compounds or extracts or culture products.

In one embodiment, the candidate factor CA is any cell, e.g., fibroblast precursors, including mesenchymal stem cells, as shown in Figures 1(D), 2(D) and 3(D). Cells, fibroblasts (eg, fibroblasts that are not damaged by light irradiation, or fibroblasts that are less damaged by light irradiation), and the like may be used. As used herein, the term "fibroblasts not damaged by light irradiation" or "fibroblasts less damaged by light irradiation" refers to fibroblasts that have not been subjected to the light irradiation step. It refers to fibroblasts with a lower level of senescence than "fibroblasts damaged by light irradiation".

By adding a candidate substance to the skin model 1, culturing for a desired period, and analyzing the pigmentation index in the skin model 1, the therapeutic or preventive effect of the candidate substance on pigmentation can be evaluated. For example, compared with the degree of pigment production and / or pigmentation of skin model 1 to which no candidate substance is added or to which any substance having no therapeutic or preventive effect on pigmentation is added, skin model 1 to which the candidate substance is added If the index of hyperpigmentation in is improved, the candidate substance can be evaluated as having therapeutic or prophylactic effect of hyperpigmentation.

Examples of "pigmentation index" include, for example, difference in amount of pigment contained, turnover level (e.g., change in expression of differentiation marker), aggregation or diffusion of pigment uptake evaluation substance, heterogeneity of cytoplasm, melanin synthesis factors (e.g., tyrosinase (TYR) in melanocytes; tyrosinase-related protein 1 (TYRP1); dopachrome tautomerase (DCT); or microphthalmia-associated transcription factor (MITF)), or endothelin 1 (ET1) in keratinocytes; or stem cells. Expression of a factor (SCF1)), enhancement of differentiation or proliferation ability (eg, Ki67), differential expression of pigment granules, enhancement of phagocytosis of keratinocytes, and the like can be used as indicators, but are not limited thereto.

As used herein, “pigmentation” refers to the production of pigments produced by melanocytes, such as melanin pigments, when the skin model 1 contains melanocytes. The amount of pigment production can be determined, for example, by extracting melanin pigment from a skin model, particularly the first cell group (keratinocytes and melanocytes), and measuring the absorbance at 405 nm. In addition, the amount of pigment production is, for example, the amount of melanin contained in the skin model, particularly the first cell group (keratinocytes and melanocytes) or the amount of nucleic acid encoding it (e.g., the amount of mRNA), ELISA method, flow cytometer method, Western blot method , immunohistochemistry, qPCR, and the like, but are not limited thereto.

As used herein, the term "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, when the amount of melanin increases, the brightness decreases, and the first cell group 10 of the skin model 1 exhibits a dark color. Conversely, when the amount of melanin decreases, the brightness increases, and the first cell group 10 of the skin model 1 exhibits a pale 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.

In one embodiment, the method of the present invention further comprises the step of adding a dye uptake evaluation substance to the first cell group before step (1), and in step (3), the dye uptake A factor for treating or preventing skin pigmentation may be evaluated by using the degree of incorporation of the evaluation substance into the first cell group as an indicator of the pigmentation (for example, FIG. 3 (A )).

In one embodiment, the method of the present invention further comprises the step of adding a dye uptake evaluation substance to the first cell group after the step (2), and in the step (3), the dye uptake A factor for treating or preventing skin pigmentation may be evaluated by using the degree of incorporation of the evaluation substance into the first cell group as an indicator of the pigmentation (for example, FIG. 2 ( F)).

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

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

Normal human epidermal melanocytes (Kurabo) were cultured in medium 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 supplemented with keratinocyte growth supplement (EDGS).

1-2. Culture of fibroblasts and PUVA treatment

Normal human fibroblasts (Cell Research Corp.) were cultured in 10% fetal bovine serum-containing Dulbecco's Modified Eagle Medium (hereinafter referred to as 10% DMEM) and seeded at 3×10 ⁵ cells in each T25 flask. Before the cell density reached 100% confluency, the cells were replaced with 10% DMEM containing a photosensitizer, psoralen (final concentration: 25 ng/mL), and cultured. After swelling with 4 mL of PBS after 24 hours, the PBS was removed and replaced with 1 mL PBS containing psoralen (final concentration 25 ng/mL), followed by 6 J/cm ² UVA irradiation (hereinafter, “PUVA treatment”). ). Then, after swelling with 4 mL of PBS, the PBS was removed and replaced with 10% DMEM, followed by culturing for 3 days, and the culture supernatant and cells were collected. The same donor-derived fibroblasts without PUVA treatment were used as a control for the PUVA-treated fibroblasts.

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

Keratinocytes or melanocytes were seeded on a 6-well plate at 1×10 ⁵ cells, respectively, and cultured for about 24 hours to allow the cells to adhere (FIG. 1(A)). Thereafter, 1×10 ⁴ cells per well of fibroblasts 3 days after PUVA treatment or untreated were seeded on the culture insert. To induce stain-like changes in epidermal cells, they were co-cultured with PUVA-treated fibroblasts for 3-5 days. When examining the difference in distribution of pigment granules in keratinocytes, fluorescent beads or pigment granules were added at the start of co-culture (Fig. 3(A)). 1/10, 1/5, 1/2, or an equivalent amount of therapeutic fibroblasts relative to the number of PUVA-treated fibroblasts initially seeded onto the culture insert after pseudostained epidermal cells were induced. (Corresponding to "non-PUVA-FB" in FIG. 1 (D), FIG. 2 (D) or FIG. 3 (D)) was added and further cultured for 3 to 5 days. We investigated the therapeutic effect. When phagocytosis in keratinocytes was examined, fluorescent beads (Polysciences Inc., Cat. #19507) or pigment granules (30a) were added at the same time as therapeutic fibroblasts (Fig. 2(F)). 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 for the culture.

1-4. Immunoantibody staining

The medium was removed from the 6-well plate of the bilayer culture model, washed with PBS, added with 4% paraformaldehyde (Wako), and allowed to stand at room temperature for about 1 hour to fix the cells. Then, after washing three times with PBS, 1 mL of a blocking agent for immunological experiments, Immunoblock (registered trademark) (DS Pharma Biomedical Co., Ltd.) was added per well and allowed to stand at room temperature for 1 hour. Then, after three washes with PBS, primary antibody Ki67 (Abcam) or P63 (Abcam) was diluted 1:100 in diluent (1% BSA-PBS) and added to fixed cells overnight at 4°C. left undisturbed. After washing with PBS three times, 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 with PBS three times, 500 μl of 0.1% Hoechst (registered trademark) 33342 (Thermo Fisher Scientific Inc.)-PBS solution was added and incubated at 37° C., 5% CO ₂ for 15 minutes to remove cell nuclei. dyed. After that, the cells were washed with PBS three times, and fluorescence observation and imaging were performed using a microscope. Immunofluorescence staining results were observed with an LSM700 laser scanning confocal microscope and images were acquired with Zen2011 software (Carl Zeiss). Representative pictures are shown as results after four independent experiments.

Cellular Senescence Detection Kit-SPiDER-βGal (Dojindo Laboratories) was used for evaluation of aging index by β-Gal according to the manufacturer's instructions. After removing the culture medium from the 6-well plate of the two-layer culture model and washing with HBSS, 1 ml of Bafilomycin A1 working solution was added, and the plate was allowed to stand in a 5% CO ₂ incubator at 37° C. for 1 hour. Subsequently, 1 ml of SPiDER-βGal working solution was added, and the mixture was allowed to stand in a 5% CO ₂ incubator at 37° C. for 30 minutes. The supernatant was removed, washed twice with HBSS, and observed under a fluorescence microscope. Representative pictures are shown as results after four independent experiments.

1-5. Fontana Masson staining (FM staining)

Tissue staining with Fontana-Masson Stain Kit (ScyTek) was used to detect melanin granules. After adding a small amount of ammonium hydroxide (Wako) dropwise to a solution obtained by diluting the Silver Solution attached to the Fontana-Masson Stain Kit 4 times with ultrapure water, it was heated to 60 ° C. and applied to keratinocytes stimulated by fibroblasts. added and allowed to stand at room temperature for 45 minutes. Thereafter, the added Silver Solution was washed away with water to remove water well, and then the Gold Chloride Solution (gold chloride solution) attached to the kit was added and reacted at room temperature for 30 seconds. Furthermore, after the Gold Chloride Solution was washed away with water, SodiumThiosulfate Solution (sodium thiosulfate) attached to the kit was added and left at room temperature for 2 minutes. Finally, after washing away the thiosulfate solution with water, nuclear staining was performed by adding the Nuclear Fast Red Solution attached to the kit and leaving it at room temperature for 10 minutes. After staining, the sample was sealed with Mount-Quick, and observed and photographed under a microscope. Image analysis software ImageJ (NIH) was used to quantify the area of melanin granules from the photographs taken.

1-6. RT-PCR

Total RNA was extracted using the 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 in three independent experiments.

1-7. Purification of the melanin-rich fraction

Normal human melanocytes were seeded in a 10-cm dish at a concentration of 2×10 ⁶ cells/dish, cultured for 24 hours, and then the medium was collected. The recovered medium was centrifuged (room temperature, 2,000×g, 10 minutes) to sediment floating melanocytes and pigment granules, and after removing the supernatant, it was resuspended in 1 ml of medium. After centrifuging the suspension again (room temperature, 20,000 × g, 5 minutes) and removing the supernatant, the resulting pellet was suspended in 1 ml of medium and placed in a hanging cell culture insert (6-well Millicell 8 mm PET) (Millipore, Billerica, Mass.) and passed through a filter to remove remaining floating cells. The collected medium was centrifuged (room temperature, 20,000×g, 5 minutes) and used as isolated melanosomes. The pellet was suspended in a small amount of medium, added to a culture medium for TLR-stimulated keratinocytes, cultured for 24 hours, and then melanosome uptake was confirmed by immunofluorescence staining.

1-8. Purification of isolated melanosomes

Confluent melanocytes were harvested and homogenized with 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). 15 passes through a 25 G needle and centrifuged (4° C., 1000×g, 10 minutes). After collecting the supernatant and re-centrifuging (4°C, 10,000 x g, 10 min), the pellet was washed twice with homogenization buffer and centrifuged again (4°C, 10,000 x g for 10 minutes). The pellet was resuspended in homogenization buffer and used immediately as the melanosome-enriched fraction.

1-9. statistical processing

Statistical processing was performed by one-dimensional analysis of variance using Graph Pad Prism 5 (Graph Pad Software, La Jolla, Calif.). When p<0.05, it was judged that there was a statistically significant difference, and the degree was indicated by the following asterisk notation. (*p<0.05, **p<0.01, ***p<0.001) All experiments were repeated at least three times to confirm reproducibility.

2. result

2-1. Differentiation/proliferation study of epidermal cells In order to confirm changes in the level of differentiation/proliferation of keratinocytes, keratinocytes co-cultured with PUVA Fb or a group of PUVA Fb plus non-irradiated Fb were used, and immunofluorescent staining was performed to identify the differentiation marker p63. Proliferation marker Ki67-positive cell ratio was calculated. As a result, p63- and Ki67-positive cells decreased in the treated group (Fig. 4), and the expression levels of the respective genes remained at about 10-20% of that in the presence of PUVA Fb alone (Fig. 5). From the above results, it was shown that non-irradiated Fb suppresses epidermal thickening caused by abnormal proliferation of keratinocytes and an increase in differentiated cells, which are often observed in spots.

2-2. Phagocytosis of Epidermal Cells To investigate the effect of co-culture with PUVA-treated fibroblasts (PUVA Fb) and therapeutic fibroblasts (non-irradiated Fb) on the phagocytosis of keratinocytes isolated from fluorescent beads and melanocytes Melanosomes were added to keratinocytes. As a result of quantification by fluorescence measurement and Fontana-Masson staining, both fluorescent beads and isolated melanosomes confirmed a dose-dependent decrease in non-irradiated Fb uptake into keratinocytes (Fig. 6). These results indicated that non-irradiated Fb inhibits phagocytosis and melanosome-specific uptake that are constantly occurring in keratinocytes.

2-3. Examination of changes in localization of melanosomes in epidermal cells In order to confirm the localization of melanosomes present in keratinocytes, an equal amount of melanosomes was incorporated into keratinocytes, and then co-cultured with PUVA Fb and a group of cells to which non-irradiated Fb was added to PUVA Fb. was performed to confirm the localization change. As a result, it was found that in the presence of PUVA Fb alone, keratinocytes, in which excessive melanosomes are diffused throughout the cytoplasm, are partially localized, causing uneven coloring (Fig. 7). On the other hand, in the non-irradiated Fb-added group, melanosomes were uniformly distributed and the formation of unevenness was suppressed.

In order to evaluate the senescence level of each keratinocyte, the protein expression of β-galactosidase and the gene expression of the senescence markers p16 and p21 were measured. Figure 9). In addition, melanosome hyperaccumulation was observed in keratinocytes with high expression of β-galactosidase.

1, 1a, 1b skin model 10 first 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 Fibroblast bFB Light-damaged fibroblast KC Keratinocyte MC Melanocyte CA Candidate substance

Claims (19)

a first cell group comprising keratinocytes and/or melanocytes seeded on the first cell culture substrate;
a second cell group containing fibroblasts arranged above the first cell group and seeded on a second cell culture substrate having a porous membrane;
A skin model comprising
The fibroblasts are fibroblasts that have been 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. The skin model according to claim 1, wherein the fibroblasts damaged by light irradiation are fibroblasts damaged by ultraviolet light irradiation in the presence of a photosensitizer. 3. 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, 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 contains 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 dye uptake. A method for manufacturing a skin model, comprising:
Above the first cell culture substratum seeded with the first cell group containing keratinocytes and/or melanocytes, the porous membrane is seeded with the second cell group containing fibroblasts damaged by light irradiation. A step of placing a second cell culture substrate and co-cultivating the first cell group and the second cell group,
wherein soluble components are exchanged between a first medium for culturing the first population of cells and a second medium for culturing the second population of cells via the porous membrane. 8. 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. 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, 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 comprises 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. A method of evaluating a factor for treating or preventing skin pigmentation, comprising:
(1) Above the first cell culture substrate seeded with the first cell group containing keratinocytes and / or melanocytes, the second cell group containing fibroblasts damaged by light irradiation is seeded Porous A step of placing a second cell culture substrate having a membrane and co-cultivating the first cell group and the second cell group to prepare a skin model,
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;
(3) analyzing the pigmentation index of the first cell group obtained in the step (2) to evaluate the therapeutic or preventive effect of the candidate factor;
A method, including The degree of uptake of the dye uptake evaluation substance into the first cell group in the step (3), further comprising the step of adding a dye uptake evaluation substance to the first cell group before the step (1). as an indicator of said pigmentation. After the step (2), the step of adding a dye uptake evaluation substance to the first cell group is further included, and in the step (3), the degree of uptake of the dye uptake evaluation substance into the first cell group is determined. 14. The method of claim 13 as an indicator of said pigmentation. The method of any one of claims 13-15, wherein said candidate factor is a fibroblast and is applied to said second cell population. 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. described method. 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, thiol-protected gold nanoclusters (AUxSRy). The method according to any one of claims 13 to 18, wherein the light irradiation is UVA light irradiation.

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