Method for evaluating the effect of promoting melanosome degradation

Abstract

To build an evaluation model close to melanosome-phagocytosing keratinocytes that actually exist in the epidermis.SOLUTION: It is possible to evaluate a melanosome degradation-promoting action and screen for a melanosome degradation-promoting substance, with high accuracy using an evaluation model obtained by culturing keratinocytes attached to one side of a liquid-permeable membrane and melanocytes attached to the other side thereof and allowing keratinocytes to phagocytose melanosomes produced by melanocytes and subsequently removing the melanocytes from the liquid-permeable membrane.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the effect of promoting melanosome degradation, a method for screening for substances that promote melanosome degradation, and a model for evaluating the effect of promoting melanosome degradation. [Background technology]

[0002] Melanosomes containing melanin produced in melanocytes are phagocytosed by keratinocytes and accumulate around the nucleus of the keratinocytes, protecting the nucleus from damage caused by ultraviolet rays. These melanosomes phagocytosed by keratinocytes do not persist permanently; they are degraded within the keratinocytes and eventually expelled from the skin through skin turnover. On the other hand, in areas with skin blemishes, melanosome production increases, but the melanosomes are not degraded and instead accumulate within keratinocytes. Therefore, promoting the degradation of melanosomes accumulated within keratinocytes is thought to be effective in improving blemishes.

[0003] Conventionally, a common method for evaluating the effect of promoting melanosome degradation involves phagocytosing melanosomes extracted from melanocytes with keratinocytes, treating them with a test substance for a certain period of time, and then measuring the amount of melanin or melanosomes (see, for example, Patent Documents 1 to 4). However, conventional evaluation methods require the use of extracted melanocytes, which has the disadvantage of increasing the work time and steps. Furthermore, conventional evaluation methods involve adding melanosomes to keratinocytes to artificially phagocytize them, and do not involve the transfer of melanosomes from melanocyte to keratinocyte. This means that the actual amount of melanosome uptake between cells cannot be reflected, and there is a risk that the cell model of melanosome-phagocytosing keratinocytes present in the epidermis cannot be accurately reproduced.

[0004] In addition, a method for evaluating the melanosome degradation-promoting effect has also been proposed in the past, in which melanocytes and keratinocytes are mixed and then a test substance is added to evaluate the melanosome degradation-promoting effect. However, in an evaluation system using such a mixed culture method, melanosome-phagocytosing keratinocytes and melanocytes are mixed, which has the disadvantage that it is not possible to evaluate the melanosome degradation-promoting effect of the test substance without excluding its effect on melanocytes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-10070 [Patent Document 2] Japanese Patent Application Publication No. 2018-70566 [Patent Document 3] Japanese Patent Application Publication No. 2018-76302 [Patent Document 4] Japanese Patent Application Publication No. 2019-218268 Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to construct an evaluation model that resembles melanosome-engulfing keratinocytes that actually exist in the epidermis. Another object of the present invention is to provide a method for evaluating the effect of promoting melanosome degradation and a method for screening for substances that promote melanosome degradation using the evaluation model. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that an evaluation model can be obtained by attaching keratinocytes to one side of a liquid-permeable membrane and melanocytes to the other side, allowing the keratinocytes to phagocytose melanosomes produced by the melanocytes, and then removing the melanocytes from the liquid-permeable membrane. This evaluation model was created using a method similar to the actual mechanism of formation of melanosome-engulfing keratinocytes in the epidermis, and that the use of this evaluation model can enable highly accurate evaluation of the melanosome degradation-promoting effect and screening of substances that promote melanosome degradation. The present invention was completed through further research based on this finding.

[0008] That is, the present invention provides the following aspects. Item 1. A method for evaluating the melanosome degradation promoting effect of a test substance, comprising: a first step in which keratinocytes are attached to one side of a liquid-permeable membrane and melanocytes are attached to the other side of the membrane, and the membrane is cultured in this state, allowing the keratinocytes to phagocytose melanosomes; a second step of removing melanocytes from the liquid-permeable membrane after the first step to prepare an evaluation model in which melanosome-phagocytosing keratinocytes are attached to one surface of the liquid-permeable membrane; a third step of incubating the evaluation model obtained in the second step in the presence of a test substance and then measuring the amount of melanin or melanosome in the keratinocytes of the evaluation model; and a fourth step of determining the melanosome degradation promoting effect of the test substance based on the amount of melanin or melanosome measured in the third step; Evaluation methods, including: Item 2. The evaluation method according to Item 1, wherein in the first step, the culture vessel is partitioned using a culture insert having a liquid-permeable membrane at the bottom to separate the keratinocytes and melanocytes and co-culture them, thereby allowing the keratinocytes to phagocytose melanosomes. Item 3. A method for screening candidate substances for substances that have an effect of promoting melanosome degradation, comprising: Step I: keratinocytes are attached to one side of a liquid-permeable membrane, and melanocytes are attached to the other side of the membrane, followed by culturing the membrane in this state, and allowing the keratinocytes to phagocytose melanosomes; Step II, after Step I, of removing melanocytes from the liquid-permeable membrane to prepare an evaluation model in which melanosome-phagocytosing keratinocytes are attached to one surface of the liquid-permeable membrane; Step III, in which the evaluation model obtained in Step II is incubated in the presence of a candidate substance, and then the amount of melanin or melanosome in the keratinocytes of the evaluation model is measured; and Step IV: selecting the candidate substance that reduced the amount of melanin or melanosome in step III as one that has the effect of promoting melanosome degradation. A screening method comprising: Item 4. The screening method according to Item 3, wherein in step I, the culture vessel is partitioned using a culture insert having a liquid-permeable membrane at the bottom to separate the keratinocytes and melanocytes and co-culture them, thereby allowing the keratinocytes to phagocytose the melanosomes. Item 5. A model for evaluating the effect of promoting melanosome degradation, Melanosome-engulfing keratinocytes are attached to one side of the liquid-permeable membrane, This evaluation model is obtained by attaching keratinocytes to one side of a liquid-permeable membrane and melanocytes to the other side, culturing the membrane in this state, allowing the keratinocytes to phagocytose melanosomes, and then removing the melanocytes from the liquid-permeable membrane. [Effects of the Invention]

[0009] In the present invention, melanosomes produced by melanocytes are transferred to keratinocytes via a liquid-permeable membrane, causing the keratinocytes to phagocytose the melanosomes. This allows the creation of an evaluation model that is similar to the melanosome-phagocytosing keratinocytes that actually exist in the epidermis, making it possible to evaluate the effect of promoting melanosome degradation and screen for substances that promote melanosome degradation with high accuracy. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of a procedure for co-culturing keratinocytes and melanocytes via a liquid-permeable membrane. [Figure 2] The keratinocyte-attached vidrigel membranes obtained in Example 1 and Comparative Examples 1 to 3 were subjected to Fontana-Masson staining, and the surface on which keratinocytes were attached was observed under an optical microscope. [Figure 3] The keratinocyte-attached vidrigel membrane obtained in Example 1 was cultured in the presence or absence of rapamycin, and then Fontana-Masson staining was performed, and the surface on which keratinocytes were attached was observed under an optical microscope. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1. Method for evaluating the effect of promoting melanosome degradation The evaluation method of the present invention is a method for evaluating the melanosome degradation-promoting activity of a test substance. Specifically, the evaluation method of the present invention is characterized by comprising the following first to fourth steps: Step 1: A step in which keratinocytes are attached to one side of a liquid-permeable membrane and melanocytes are attached to the other side, and the membrane is cultured to allow the keratinocytes to phagocytose melanosomes. Second step: After the first step, the melanocytes are removed from the liquid-permeable membrane to prepare an evaluation model in which melanosome-phagocytosing keratinocytes are attached to one side of the liquid-permeable membrane. The third step: a step of incubating the evaluation model obtained in the second step in the presence of a test substance, and then measuring the amount of melanin or melanosome in the keratinocytes of the evaluation model. Step 4: A step of determining the melanosome degradation promoting effect of the test substance based on the amount of melanin or melanosome measured in step 3.

[0012] The evaluation method of the present invention will be explained below step by step.

[0013] [1st step] In the first step, keratinocytes are attached to one side of a liquid-permeable membrane, and melanocytes are attached to the other side and cultured, allowing the keratinocytes to phagocytose melanosomes. In the first step, melanosomes produced by melanocytes are transferred to keratinocytes via the liquid-permeable membrane, generating melanosome-phagocytic keratinocytes.

[0014] A liquid-permeable membrane is a membrane that is permeable to the culture medium and can serve as a support for keratinocytes and melanocytes. The type of liquid-permeable membrane is not particularly limited, but a porous membrane is preferred. Examples of materials for the porous membrane include collagen, polycarbonate, polyester (polyethylene terephthalate, etc.), polystyrene, and polytetrafluoroethylene. Among these, collagen is preferred. The pore size of the porous membrane is not particularly limited, as long as it is impermeable to cells and permeable to the culture medium, but may be, for example, 10 to 2000 nm, preferably 30 to 1500 nm, and more preferably 50 to 1000 nm.

[0015] Furthermore, the origin of the keratinocytes and melanocytes used in the first step is not particularly limited, but from the viewpoint of obtaining an evaluation model suitable for evaluating the melanosome degradation-promoting effect in humans and screening melanosome degradation-promoting substances applicable to humans, it is preferable that they are of human origin.

[0016] In the first step, keratinocytes are attached to one side of a liquid-permeable membrane and melanocytes are attached to the other side, and the membrane is cultured in this state, for example, by the following steps 1-1 and 1-2.

[0017] Step 1-1 Either keratinocytes or melanocytes are seeded in a culture vessel with a liquid-permeable membrane set therein and primary culture is carried out, thereby attaching the keratinocytes or melanocytes to one side of the liquid-permeable membrane. Step 1-2: After step 1-1, the liquid-permeable membrane is placed in a culture vessel with the side to which the keratinocytes or melanocytes are not attached facing up, and the other cell type (keratinocytes or melanocytes) is seeded and co-cultured to allow the keratinocytes to phagocytose the melanosomes.

[0018] The culture vessel used in step 1-1 may be any vessel that allows cell culture on a liquid-permeable membrane, and examples thereof include a culture plate, a culture dish, etc. A preferred example of the culture vessel used in step 1-1 is a culture plate.

[0019] The cells seeded in step 1-1 and step 1-2 may be keratinocytes on one hand and melanocytes on the other hand. For example, melanocytes may be seeded in step 1-1 and keratinocytes may be seeded in step 1-2, or keratinocytes may be seeded in step 1-1 and melanocytes in step 1-2, but it is preferable to seed melanocytes in step 1-1 and keratinocytes in step 1-2.

[0020] In step 1-1, the number of cells to be seeded on the liquid-permeable membrane is not particularly limited. For example, 1 × 10 2 ~1×10 5 cells / cm 2 Approximately, preferably 5 x 10 2 ~8×10 4 cells / cm 2 approximately, more preferably 1×10 3 ~4×10 4 cells / cm 2 The degree of

[0021] The medium used for the primary culture in step 1-1 may be appropriately selected depending on the type of cells to be seeded, and examples thereof include Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, and 199 medium. The medium used for the primary culture may contain bovine serum albumin (BSA), fetal calf serum (FCS), etc., as needed. Furthermore, media optimized for melanocyte culture (e.g., "DermaLife M Comp kit," Life Cell Technology) and media optimized for keratinocyte culture (e.g., "HUMEDIA-KG2," Kurabo Industries, Ltd.) are commercially available, and commercially available dedicated media can also be used.

[0022] The culture time for the primary culture in step 1-1 may be appropriately set within a range that allows the cells to adhere to the liquid-permeable membrane depending on the type of cells to be seeded, the number of cells to be seeded, etc., and may be, for example, 12 to 96 hours, preferably 18 to 82 hours, and more preferably 24 to 48 hours. During the culture period, the medium may be replaced with a fresh medium as needed.

[0023] The culture conditions for the primary culture in step 1-1 may be set to conditions that allow the cells to be seeded to grow, for example, in an approximately 5% CO2 environment at 36 to 38°C, preferably 37°C.

[0024] The culture vessel used in step 1-2 may be any vessel that allows cell culture on a liquid-permeable membrane, and the culture vessel used in step 1-1 can be subsequently used.

[0025] In step 1-2, the liquid-permeable membrane after step 1-1 is placed in a culture vessel with the side to which cells are attached facing downward and the side to which cells are not attached facing upward. In this state, the keratinocytes or melanocytes that were not seeded in step 1-1 are seeded on the side of the liquid-permeable membrane to which cells are not attached (the upper surface of the liquid-permeable membrane in the culture vessel), and the keratinocytes are co-cultured while separated by the liquid-permeable membrane.

[0026] In step 1-2, the number of cells to be seeded on the liquid-permeable membrane is not particularly limited. For example, 1 × 10 2 ~1×10 5 cells / cm 2 Approximately, preferably 5 x 10 2 ~8×10 4 cells / cm 2 approximately, more preferably 1×10 3 ~4×10 4 cells / cm 2 The degree of

[0027] The medium used for the co-culture in step 1-2 may be any medium capable of growing both keratinocytes and melanocytes, such as Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, and 199 medium. The medium used for the co-culture may contain bovine serum albumin (BSA), fetal calf serum (FCS), or the like, as needed. Furthermore, as long as both keratinocytes and melanocytes can grow in the medium, a medium optimized for keratinocyte culture (e.g., "HUMEDIA-KG2," Kurabo Industries, Ltd.) may also be used.

[0028] The culture time for the co-culture in step 1-2 may be set within a range in which melanosomes in the melanocytes are transferred to keratinocytes and melanosome-engulfing keratinocytes are produced, for example, 2 to 10 days, preferably 3 to 9 days, and more preferably 3 to 8 days. During the culture period, the medium may be replaced with a fresh medium as needed.

[0029] Furthermore, the culture atmosphere for the co-culture in step 1-2 may be set to conditions that allow the keratinocytes and melanocytes to grow, for example, in an atmosphere of approximately 5% CO2 at 36 to 38°C, preferably 37°C.

[0030] Thus, by carrying out step 1-2, a liquid-permeable membrane is obtained in which melanosome-phagocytosing keratinocytes are attached to one surface and melanocytes are attached to the other surface.

[0031] Steps 1-1 and 1-2 can be performed by using a culture insert with a liquid-permeable membrane at the bottom to separate the culture vessel and co-culture the keratinocytes and melanocytes. The procedure for performing steps 1-1 and 1-2 using this culture vessel is shown in (1) and (2) below, and a schematic diagram is shown in Figure 1. Note that, for convenience, Figure 1 shows keratinocytes attached to the liquid-permeable membrane on the inner surface of the culture insert and melanocytes attached to the liquid-permeable membrane on the outer surface of the culture insert, but the arrangement of keratinocytes and melanocytes may be reversed. (1) First, the porous membrane on the outer surface of the culture insert is placed on top, and a ring-shaped holder is attached that extends upward from the outer periphery of the outer surface of the porous membrane to form a container, and a suspension of one type of cell is placed in the container and cultured, thereby causing the cell to adhere to the porous membrane on the outer surface of the culture insert (step 1-1). (2) Next, the culture medium is removed, the ring-shaped holder is removed, the culture insert is attached to a plate (companion plate) that serves as a receptacle for the culture insert, and a suspension of the other cell is placed in the culture insert and cultured (step 1-2).

[0032] [Second process] In the second step, the melanocytes attached to the liquid-permeable membrane after the first step are removed to prepare an evaluation model in which melanosome-phagocytosing keratinocytes are attached to one side of the liquid-permeable membrane. By removing the melanocytes attached to one side of the liquid-permeable membrane in the second step, it becomes possible to evaluate the melanosome degradation-promoting effect without the influence of melanocytes.

[0033] In the second step, it is necessary to remove only the melanocytes present on one side of the liquid-permeable membrane after the first step, while the melanosome-engulfing keratinocytes present on the other side must remain attached to the liquid-permeable membrane. Therefore, it is preferable not to use chemical treatment such as trypsin to remove the melanocytes, but to perform the removal physically using, for example, a cotton swab or spatula.

[0034] [3rd step] In the third step, the evaluation model is incubated in the presence of a test substance, and then the amount of melanin in the keratinocytes of the evaluation model is measured.

[0035] A test substance is a substance to be evaluated for its ability to promote melanosome degradation. The test substance may be a substance known to promote melanosome degradation, or it may be a substance whose activity is unknown. The type of test substance is not particularly limited, and examples include natural products, synthetic products, and semi-synthetic products. Specific examples of test substances include proteins, peptides, amino acids, plant extracts, animal extracts, sugars (including polysaccharides, oligosaccharides, monosaccharides, etc.), lipids (including phospholipids, glycolipids, etc.), surfactants, other high molecular weight organic compounds, other low molecular weight organic compounds, inorganic compounds, other biological compounds, modified products thereof, and derivatives thereof. The test substance may consist of only one compound or may contain two or more compounds.

[0036] In the third step, the concentration of the test substance during incubation is not particularly limited, but may be set to, for example, about 0.01 to 2.0% by weight. The concentration of the test substance may also be set in stages from low to high.

[0037] Furthermore, in the third step, the solvent used when the test substance and the evaluation model are coexisted may be any solvent that can maintain the viability of keratinocytes, such as culture medium, buffer solution, and physiological saline. Among these, culture medium is preferred. When a culture medium is used as the solvent, the type of medium is not particularly limited, and examples include Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, and 199 medium. Furthermore, the medium may contain bovine serum albumin (BSA), fetal calf serum (FCS), etc., as needed. Alternatively, a medium optimized for keratinocyte culture (e.g., "HUMEDIA-KG2", Kurabo Industries, Ltd.) can also be used.

[0038] The incubation time in the third step may be appropriately set depending on the type of candidate substance, etc., and may be, for example, 1 to 5 days, preferably 2 to 5 days, more preferably 2 to 3 days. During the incubation period, the solvent may be exchanged with fresh solvent containing the candidate substance, if necessary.

[0039] The incubation atmosphere in the third step may be set to conditions that allow the keratinocytes and melanocytes to remain viable, for example, at 36 to 38°C, preferably 37°C, in an atmosphere of approximately 5% CO2.

[0040] In the third step, after incubation in the presence of the test substance, the amount of melanin or melanosome in the keratinocytes of the evaluation model is measured.

[0041] The amount of melanin in keratinocytes can be qualitatively measured, for example, by Fontana-Masson staining, or quantified by extracting melanin from keratinocytes with 1N sodium hydroxide solution and measuring the absorbance of the resulting extract at 450 nm.

[0042] Furthermore, the amount of melanosomes in keratinocytes can be quantitatively or qualitatively measured by a method for measuring the amount of melanosome membrane proteins such as Pmel17, gp87, etc. Specifically, the amount of melanosome membrane proteins can be measured by immunostaining, Western blotting, etc.

[0043] [4th step] In the fourth step, the melanosome degradation-promoting effect of the test substance is determined based on the amount of melanin or melanosome measured in the third step. Specifically, a test substance that reduces the amount of melanin or melanosome in the third step is determined to have the effect of promoting melanosome degradation. On the other hand, a test substance that does not reduce the amount of melanin or melanosome in the third step is determined to have no effect of promoting melanosome degradation.

[0044] In the fourth step, whether or not the test substance reduces the amount of melanin or melanosomes can be determined by comparing it with a control to which the third step was performed without adding the test substance. Specifically, the lower the amount of melanin or melanosomes compared to the control, the greater the effect of the test substance in promoting melanosome degradation. Furthermore, a test substance that has the same amount of melanin or melanosomes as the control can be determined to have no effect in promoting melanosome degradation.

[0045] 2. Screening method for substances that promote melanosome degradation The screening method of the present invention is a method for selecting a substance that has the effect of promoting melanosome degradation from among candidate substances. Specifically, the screening method of the present invention is characterized by comprising the following steps I to IV. Step I: A step in which keratinocytes are attached to one side of a liquid-permeable membrane and melanocytes are attached to the other side, followed by culturing, and allowing the keratinocytes to phagocytose melanosomes. Step II: After step I, the melanocytes are removed from the liquid-permeable membrane to prepare an evaluation model in which melanosome-phagocytosing keratinocytes are attached to one side of the liquid-permeable membrane. Step III: A step of incubating the evaluation model obtained in Step II in the presence of a candidate substance, and then measuring the amount of melanin or melanosome in the keratinocytes of the evaluation model. Step IV: A step of selecting a candidate substance that reduced the amount of melanin or melanosome in step III as one that has the effect of promoting the degradation of melanosomes.

[0046] Each step of the screening method of the present invention will be explained below.

[0047] [Step I and Step II] Steps I and II are the same as steps 1 and 2, respectively, in the evaluation method.

[0048] [Step III] In step III, the evaluation model is incubated in the presence of a candidate substance, and then the amount of melanin or melanosome in the keratinocytes of the evaluation model is measured.

[0049] A candidate substance is a substance that is subjected to screening to determine whether or not it has the effect of promoting melanosome degradation. The type of candidate substance is not particularly limited, and examples include natural products, synthetic substances, and semi-synthetic substances. Specific examples of candidate substances include proteins, peptides, amino acids, plant extracts, animal extracts, sugars (including polysaccharides, oligosaccharides, monosaccharides, etc.), lipids (including phospholipids, glycolipids, etc.), surfactants, other high molecular weight organic compounds, other low molecular weight organic compounds, inorganic compounds, other biological compounds, modified products thereof, and derivatives thereof. A candidate substance may consist of only one type of compound, or may contain two or more types of compounds.

[0050] In step III, the concentration of the candidate substance during incubation is the same as the concentration of the test substance in step 3 of the screening method. In addition, in step III, the solvent used, incubation time, incubation atmosphere, and method for measuring the amount of melanin or melanosome after incubation are the same as those in step 3 of the screening method.

[0051] [Step IV] In step IV, candidate substances that have reduced the amount of melanin or melanosomes in step III are selected as those that have the effect of promoting the degradation of melanosomes.

[0052] In step III, whether or not the amount of melanin or melanosomes is reduced can be determined by comparing with a control in which step III was carried out without adding the test substance. Specifically, if the amount of melanin or melanosomes is lower than that of the control, the candidate substance is determined to have an effect of promoting melanosome degradation, and the lower the amount of melanin or melanosomes is compared to that of the control, the stronger the effect of the candidate substance in promoting melanosome degradation can be determined.

[0053] Candidate substances selected as those having the effect of promoting melanosome degradation are expected to be used in the field of external preparations such as cosmetics as ingredients expected to have whitening effects, etc. In addition, candidate substances selected as those having the effect of promoting melanosome degradation can also be used as experimental tools for elucidating the mechanism of melanosome degradation, etc.

[0054] 3. Model for evaluating the effect of promoting melanosome degradation The evaluation model of the present invention is an evaluation model used for evaluating the effect of promoting melanosome degradation, and is characterized in that melanosome-phagocytosing keratinocytes are attached to one surface of a liquid-permeable membrane, and the model is obtained by culturing the liquid-permeable membrane with keratinocytes attached to one surface and melanocytes attached to the other surface, thereby allowing the keratinocytes to phagocytose the melanosomes, and then removing the melanocytes from the liquid-permeable membrane. The evaluation model of the present invention will be described in detail below.

[0055] The evaluation model of the present invention is a cell-attached membrane in which melanosome-phagocytic keratinocytes are attached to one side of the liquid-permeable membrane and no cells are attached to the other side of the liquid-permeable membrane. Specifically, the evaluation model of the present invention is obtained through the first and second steps of the evaluation method.

[0056] In the evaluation model of the present invention, the melanosome-phagocytic keratinocytes attached to the liquid-permeable membrane are prepared using a method similar to the mechanism by which melanosome-phagocytic keratinocytes are formed in the actual epidermis, and therefore can be used as model cells for evaluating the effect of promoting melanosome degradation and screening substances that promote melanosome degradation. [Example]

[0057] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0058] Test Example 1 1. Preparation of a liquid-permeable membrane with attached keratinocytes [Example 1] A liquid-permeable membrane with melanosome-engulfing keratinocytes attached was prepared using Ad-MED Vidrigel 2 (12-well type, manufactured by Kanto Chemical Co., Inc.). The ad-MED Vidrigel 2 contained a Vidrigel membrane (a porous membrane with densely folded collagen fibers, membrane area 1 cm). 2The test kit was equipped with a culture insert with a bottom containing micropores (pore size 67 nm) and a 12-well plate to hold the culture insert. An optional ring (ring-shaped holder) purchased as an accessory for the ad-MED Vidrigel 2 was also used. The optional ring was attached to the culture insert with the Vidrigel membrane facing upward, extending upward from the periphery of the outer surface of the Vidrigel membrane, to temporarily form wells.

[0059] First, an optional ring was attached to the culture insert, and the Vidrigel membrane on the outer surface of the culture insert formed the bottom and the optional ring formed the wall of a well. Next, 4 × 10 human normal melanocytes (NHEM (NB), Kurabo) suspended in DermaLife M Comp kit medium (Life Cell Technology) were added. 4 The cells were seeded into wells with walls formed with an optional ring so that the number of cells per well was 1, and the cells were cultured for 2 days in an environment of 37°C and 5% CO2, allowing the melanocytes to attach to the vidrigel membrane on the outer surface of the culture insert.

[0060] Next, the culture medium was removed from the wells, and the option ring was removed. The culture insert with melanocytes attached to the vidrigel membrane on the outer surface (lower side) was inserted into the well of a 12-well plate containing HUMEDIA-KG2 medium (Kurabo Industries, Ltd.) and fixed. Then, 1 × 10 human normal keratinocytes (NHEK (NB), Kurabo Industries, Ltd.) suspended in HUMEDIA-KG2 medium were added to the culture insert. 4 The cells were seeded at 10 cells / well and cultured at 37°C in an environment of 5% CO2 for 7 days.

[0061] Next, the culture insert was removed from the well of the 12-well plate, and melanocytes attached to the vidrigel membrane on the outer surface of the culture insert were removed using a sterilized cotton swab soaked in PBS(-), yielding a keratinocyte-attached vidrigel membrane.

[0062] [Comparative Example 1] Keratinocytes were cultured in a culture insert using Ad-MED Vidrigel 2 (12-well type, manufactured by Kanto Chemical Co., Inc.) to obtain a keratinocyte-attached Vidrigel membrane. The keratinocyte culture conditions were the same as those in Example 1.

[0063] Comparative Example 2 HAMF12 medium containing 10% FBS (fetal bovine serum) was used to culture 75 cm 2 Human melanoma cells (HMV-II, RCB368) were cultured in a flask at 37°C in a 5% CO2 atmosphere until confluent. After incubation, the cells were harvested and centrifuged. The supernatant was discarded, and the cell pellet was frozen at -20°C. The frozen cell pellet was thoroughly suspended in 1 ml of lysis buffer (0.1 M Tris-HCl, pH 7.5, 1% Igepal CA-630, 0.01% SDS) and allowed to stand at 4°C for 20 minutes. After incubation, the cell lysate was centrifuged at 1,000 × g for 5 minutes at 4°C. The resulting supernatant was transferred to a microtube and centrifuged at 1,000 × g for 5 minutes at 4°C. The supernatant was transferred to a new microtube and centrifuged at 20,000 × g for 5 minutes at 4°C. The resulting pellet was then centrifuged at 20,000 × g for 5 minutes at 4°C. The resulting supernatant was removed. A melanosome solution was prepared by adding 0.05 ml of PBS(-) to the pellet.

[0064] HUMEDIA-KG2 medium (Kurabo Industries, Ltd.) was added to the wells of a 12-well plate of Ad-MED Vidrigel 2 (12-well type, Kanto Chemical Co., Ltd.), and then a culture insert was inserted and fixed. Then, 1 × 10 human normal keratinocytes (NHEK (NB), Kurabo Industries, Ltd.) suspended in HUMEDIA-KG2 medium were placed in the culture insert. 4 50 μl of the melanosome solution prepared above was added, and the mixture was cultured at 37°C in 5% CO for 7 days. Thus, a keratinocyte-attached vidrigel membrane incorporating melanosomes was obtained.

[0065] [Comparative Example 3] A keratinocyte-attached vidrigel membrane was obtained under the same conditions as in Comparative Example 2, except that the extracted melanosomes were not added.

[0066] 2. Evaluation of the liquid-permeable membrane to which keratinocytes are attached The keratinocyte-attached vidrigel membranes obtained in Example 1 and Comparative Examples 1 to 3 were stained with Fontana-Masson staining to visualize melanin, and the surface to which the keratinocytes were attached was observed under an optical microscope. The results are shown in Figure 2. As a result, in Comparative Example 1, in which melanosomes were extracted and added to keratinocytes, melanosome phagocytosis was confirmed only in some keratinocytes, whereas in Example 1, in which keratinocytes and melanocytes were co-cultured while separated by a vidrigel membrane, melanosome phagocytosis was confirmed uniformly throughout the keratinocytes.

[0067] Test Example 2 Under the same conditions as in Example 1, a culture insert having a keratinocyte-attached vidrigel membrane (melanocytes had been removed) was prepared.

[0068] The culture inserts were inserted into wells of a 12-well plate containing HUMEDIA-KG2 medium and fixed. Then, 0.5 ml of HUMEDIA-KG2 medium containing 1 μg / ml of rapamycin was added to the culture insert, and the cells were cultured for 3 days at 37°C in a 5% CO atmosphere. As a control, the cells were cultured under the same conditions as above, except that HUMEDIA-KG2 medium containing no rapamycin was used instead of the HUMEDIA-KG2 medium containing 1 μg / ml of rapamycin. Rapamycin is a compound that has been suggested to have melanosome-degrading properties.

[0069] After culturing, the cell-attached membrane was stained with Fontana-Masson staining to visualize melanin, and the surface on which keratinocytes were attached was observed under an optical microscope.

[0070] Furthermore, after culturing, the cells were detached from the cell adhesion membrane and 1 × 10 5The cells were immersed in 0.1 ml of 1N aqueous sodium hydroxide solution and disrupted by ultrasound, then thoroughly stirred and incubated at 80°C for 20 minutes to extract melanin. The absorbance at 450 nm was measured to determine the amount of melanin in the keratinocytes.

[0071] The results of Fontana-Masson staining and observation under an optical microscope are shown in Figure 3, and the results of measuring the amount of melanin in the keratinocytes are shown in Table 1. As a result, when cultured with the addition of rapamycin, the amount of melanin was found to be reduced compared to the control, confirming that the keratinocyte-attached vidrigel membrane prepared in Example 1 can be used to correctly evaluate melanosome degradation activity.

[0072] [Table 1]

Claims

1. A method for evaluating the melanosome degradation promoting effect of a test substance, comprising: a first step of attaching keratinocytes to one surface of a liquid-permeable membrane and melanocytes to the other surface of the membrane, culturing the membrane in this state, and allowing the keratinocytes to phagocytose melanosomes; a second step of removing melanocytes from the liquid-permeable membrane after the first step to prepare an evaluation model in which melanosome-phagocytosing keratinocytes are attached to one surface of the liquid-permeable membrane; a third step of incubating the evaluation model obtained in the second step in the presence of a test substance and then measuring the amount of melanin or melanosome in the keratinocytes of the evaluation model; and A fourth step of determining the melanosome degradation promoting effect of the test substance based on the amount of melanin or melanosome measured in the third step. Evaluation methods, including:

2. The evaluation method according to claim 1, wherein in the first step, the culture vessel is partitioned using a culture insert having a liquid-permeable membrane at the bottom, and the keratinocytes and melanocytes are co-cultured at a distance, thereby allowing the keratinocytes to phagocytose the melanosomes.

3. A method for screening candidate substances for a substance that has an effect of promoting melanosome degradation, comprising the steps of: Step I: keratinocytes are attached to one surface of a liquid-permeable membrane, and melanocytes are attached to the other surface of the membrane, followed by culturing the membrane in this state, and allowing the keratinocytes to phagocytose melanosomes; Step II, after Step I, of removing melanocytes from the liquid-permeable membrane to prepare an evaluation model in which melanosome-phagocytosing keratinocytes are attached to one surface of the liquid-permeable membrane; Step III, in which the evaluation model obtained in step II is incubated in the presence of a candidate substance, and then the amount of melanin or melanosome in the keratinocytes of the evaluation model is measured; and Step IV: selecting the candidate substance that reduced the amount of melanin or melanosome in Step III as one that has the effect of promoting melanosome degradation. A screening method comprising:

4. The screening method according to claim 3, wherein in step I, the culture vessel is partitioned using a culture insert having a liquid-permeable membrane at the bottom to separate and co-culture keratinocytes and melanocytes, thereby allowing the keratinocytes to phagocytose melanosomes.

5. Use of an evaluation model for evaluating the melanosome degradation promoting effect of a test substance, comprising: the evaluation model has melanosome-phagocytosing keratinocytes attached to one surface of a liquid-permeable membrane, and is obtained by culturing the liquid-permeable membrane in a state in which the keratinocytes are attached to one surface and the melanocytes are attached to the other surface, thereby causing the keratinocytes to phagocytose the melanosomes, and then removing the melanocytes from the liquid-permeable membrane; The above use, wherein the evaluation model is incubated in the presence of a test substance.

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