JP7748799B2 - Method for evaluating the effect of promoting melanosome degradation - Google Patents

Description

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

Melanosomes containing melanin produced within melanocytes are phagocytosed by keratinocytes and accumulate around the nucleus within the keratinocytes, protecting the nucleus from damage caused by ultraviolet rays. These melanosomes phagocytosed by keratinocytes do not persist permanently; they are broken down 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 broken down and instead accumulate within keratinocytes. Therefore, promoting the breakdown of melanosomes accumulated within keratinocytes is thought to be effective in improving blemishes.

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 measuring the amount of melanin or melanosomes (see, for example, Patent Documents 1 to 4). However, because conventional evaluation methods require the use of extracted melanocytes, they have the disadvantage of increasing work time and steps. Furthermore, conventional evaluation methods involve adding melanosomes to keratinocytes to artificially phagocytize them, and because melanosomes are not transferred from melanocyte to keratinocyte, they do not reflect the actual amount of melanosome uptake between cells, and there is a risk that they do not accurately reproduce the cell model of melanosome-phagocytosing keratinocytes present in the epidermis.

In addition, a method proposed for evaluating the melanosome degradation-promoting effect has been to culture melanocytes and keratinocytes together, then add a test substance to evaluate the melanosome degradation-promoting effect. However, this type of evaluation system using a mixed culture method has the disadvantage that melanosome-phagocytosing keratinocytes and melanocytes are present together, making it impossible to evaluate the melanosome degradation-promoting effect of the test substance without excluding its effect on melanocytes.

JP 2015-10070 A Japanese Patent Application Laid-Open No. 2018-70566 Japanese Patent Application Laid-Open No. 2018-76302 Japanese Patent Application Laid-Open No. 2019-218268

One object of the present invention is to construct an evaluation model that closely resembles melanosome-engulfing keratinocytes that actually exist in the epidermis. Another object of the present invention is to use this evaluation model to provide a method for evaluating the effect of promoting melanosome degradation and a method for screening for substances that promote melanosome degradation.

The inventors conducted extensive research to solve the above-mentioned problems and found that an evaluation model was created 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 the 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 found that the use of this evaluation model makes it possible to evaluate the effect of promoting melanosome degradation and screen for substances that promote melanosome degradation with high accuracy. The present invention was completed through further research based on these findings.

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 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;
The evaluation method includes 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 melanosomes in the keratinocytes of the evaluation model; and a fourth step of determining the effect of the test substance on promoting the degradation of melanosomes based on the amount of melanin or melanosomes measured in the third step.
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 a substance that has an effect of promoting melanosome degradation, comprising:
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;
A screening method comprising: step III of incubating the evaluation model obtained in step II in the presence of a candidate substance, and then measuring the amount of melanin or melanosomes in the keratinocytes of the evaluation model; and step IV of selecting a candidate substance that reduces the amount of melanin or melanosomes in step III as one that has the effect of promoting melanosome degradation.
Item 4. The screening method according to Item 3, wherein in step I, the keratinocytes and melanocytes are co-cultured at a distance by dividing the culture vessel using a culture insert having a liquid-permeable membrane at the bottom, thereby allowing the keratinocytes to phagocytose melanosomes.
Item 5. A model for evaluating the effect of promoting melanosome degradation, comprising:
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.

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 closely resembles 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.

FIG. 1 is a diagram schematically illustrating an example of a procedure for co-culturing keratinocytes and melanocytes via a liquid-permeable membrane. 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. FIG. 1 shows images of the surface of the keratinocyte-attached vidrigel membrane obtained in Example 1, which was cultured in the presence or absence of rapamycin, and then stained with Fontana-Masson, and observed under an optical microscope.

1. Method for evaluating the effect of promoting melanosome degradation The evaluation method of the present invention is a method for evaluating the effect of a test substance of promoting melanosome degradation. Specifically, the evaluation method of the present invention is characterized by comprising the following steps 1 to 4:
First step: A step of attaching keratinocytes to one side of a liquid-permeable membrane and culturing melanocytes attached to the other side, and allowing 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.
Step 3: A step of incubating the evaluation model obtained in step 2 in the presence of a test substance, and then measuring the amount of melanin or melanosomes 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.

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

[First step]
In the first step, keratinocytes are attached to one side of a liquid-permeable membrane, and melanocytes are attached to the other side. These are then cultured, and melanosomes are phagocytosed by the keratinocytes. In the first step, melanosomes produced by the melanocytes are transferred to the keratinocytes via the liquid-permeable membrane, generating melanosome-phagocytosing keratinocytes.

A liquid-permeable membrane is a membrane that is permeable to the culture medium and can serve as a support for keratinocytes and melanocytes. There are no particular limitations on the type of liquid-permeable membrane, but a porous membrane is preferred. Examples of materials for porous membranes include collagen, polycarbonate, polyester (polyethylene terephthalate, etc.), polystyrene, and polytetrafluoroethylene. Of these, collagen is preferred. There are also no particular limitations on the pore size of the porous membrane, as long as it is impermeable to cells and permeable to the culture medium. Examples of the pore size include 10 to 2000 nm, preferably 30 to 1500 nm, and more preferably 50 to 1000 nm.

The origin of the keratinocytes and melanocytes used in the first step is not particularly limited, but from the perspective 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 be of human origin.

In the first step, keratinocytes are attached to one side of the liquid-permeable membrane, and melanocytes are attached to the other side, and the cells are cultured in this state. This can be done, for example, by the following steps 1-1 and 1-2.

Step 1-1 : Either keratinocytes or melanocytes are seeded into a culture vessel in which a liquid-permeable membrane has been set 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.

The culture vessel used in step 1-1 may be any vessel that allows cell culture on a liquid-permeable membrane, such as a culture plate or culture dish. A preferred culture vessel used in step 1-1 is a culture plate.

The cells seeded in steps 1-1 and 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.

In step 1-1, the number of cells to be seeded on the liquid-permeable membrane is not particularly limited, but may be, for example, ^about 1 x ¹⁰ to 1 x ¹⁰ cells/cm, preferably about 5 x ¹⁰ to ⁸ x ¹⁰ cells/cm, and more preferably about 1 x ¹⁰ to 4 x ¹⁰ cells/cm ^.

The medium used for the primary culture in step 1-1 may be selected appropriately depending on the type of cells to be seeded, and examples 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.

The culture time for the primary culture in step 1-1 may be set appropriately depending on the type of cells to be seeded, the number of cells to be seeded, etc., as long as the cells can adhere to the liquid-permeable membrane. Examples of the culture time include 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 fresh medium as needed.

The culture conditions for the primary culture in step 1-1 may be set so that the cells to be seeded can grow, for example, at 36 to 38°C, preferably 37°C, in an environment of about 5% _CO2 .

The culture vessel used in step 1-2 can be any vessel that allows cell culture on a liquid-permeable membrane, and the culture vessel used in step 1-1 can continue to be used.

In step 1-2, the liquid-permeable membrane from step 1-1 is placed in a culture vessel with the side with cells attached facing downwards and the side without cells facing upwards. 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 side of the liquid-permeable membrane in the culture vessel), and the keratinocytes are co-cultured while separated by the liquid-permeable membrane.

In step 1-2, the number of cells to be seeded on the liquid-permeable membrane is not particularly limited, but may be, for example, ^about 1 x ¹⁰ to 1 x ¹⁰ cells/cm, preferably about 5 x ¹⁰ to ⁸ x ¹⁰ cells/cm, and more preferably about 1 x ¹⁰ to 4 x ¹⁰ cells/cm ^.

The medium used for co-culture in step 1-2 may be any medium capable of supporting the growth of 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 co-culture may also contain bovine serum albumin (BSA), fetal calf serum (FCS), etc., as needed. Furthermore, as long as both keratinocytes and melanocytes can grow in it, a medium optimized for keratinocyte culture (e.g., "HUMEDIA-KG2," Kurabo Industries, Ltd.) can also be used.

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 fresh medium as needed.

The culture atmosphere for the co-culture in step 1-2 may be set to conditions that allow the growth of keratinocytes and melanocytes, for example, at 36 to 38°C, preferably 37°C, in an atmosphere of approximately 5% _CO2 .

Thus, by carrying out steps 1-2, a liquid-permeable membrane is obtained with melanosome-phagocytosed keratinocytes attached to one side and melanocytes attached to the other side.

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).

[Second step]
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.

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

[Third 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.

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 for which this effect is unknown. The type of test substance is not particularly limited, and examples include natural products, synthetic substances, and semi-synthetic substances. 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 type of compound, or it may contain two or more types of compounds.

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

In addition, in the third step, the solvent used to coexist the test substance and the evaluation model may be any solvent that can maintain the viability of keratinocytes, such as culture medium, buffer solution, or physiological saline. Among these, culture medium is preferred. When using culture medium 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.

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

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 .

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

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

In addition, the amount of melanosomes in keratinocytes can be measured quantitatively or qualitatively by measuring the amount of melanosome membrane proteins such as Pmel17 and gp87. Specifically, the amount of melanosome membrane proteins can be measured by immunostaining, Western blotting, etc.

[Fourth step]
In step 4, the melanosome degradation-promoting effect of the test substance is determined based on the amount of melanin or melanosome measured in step 3. Specifically, a test substance that reduces the amount of melanin or melanosome in step 3 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 step 3 is determined to have no effect of promoting melanosome degradation.

In step 4, whether the test substance reduces the amount of melanin or melanosomes can be determined by comparing it with a control to which step 3 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.

2. Screening Method for Substances Promoting 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 of attaching keratinocytes to one surface of a liquid-permeable membrane and culturing melanocytes attached to the other surface, thereby 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 melanosome degradation.

The screening method of the present invention will be explained step by step below.

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

[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.

A candidate substance is a substance that is subjected to screening to determine whether or not it has the effect of promoting melanosome degradation. There are no particular limitations on the type of candidate substance, 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.

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. Furthermore, in step III, the solvent used, incubation time, incubation atmosphere, and method for measuring the amount of melanin or melanosomes after incubation are the same as those in step 3 of the screening method.

[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.

Whether or not the amount of melanin or melanosomes has been reduced in Step III 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 the effect of promoting melanosome degradation, and the lower the amount of melanin or melanosomes is compared to the control, the stronger the effect of the candidate substance in promoting melanosome degradation.

Candidate substances selected as those with the ability to promote melanosome degradation are expected to be used in external preparations such as cosmetics as ingredients with potential for skin-whitening effects. Candidate substances selected as those with the ability to promote melanosome degradation can also be used as experimental tools to elucidate the mechanisms of melanosome degradation.

3. Evaluation Model for 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 is described in detail below.

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 steps 1 and 2 of the evaluation method described above.

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

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

Test Example 1
1. Preparation of a liquid-permeable membrane with keratinocytes attached [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 was equipped with a culture insert with a vidrigel membrane (a porous membrane composed of densely folded collagen fibers, with a membrane area of 1 ^cm2 and a pore diameter of 67 nm) at the bottom and a 12-well plate that served as a receptacle for 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, thereby temporarily forming wells.

First, an option ring was attached to the culture insert, and the vidrigel membrane on the outer surface of the culture insert formed the bottom of a well, with the option ring forming the wall. Next, human normal melanocytes (NHEM(NB), Kurabo Industries, Ltd.) suspended in DermaLife M Comp kit medium (Life Cell Technology) were seeded into the well with the wall formed by the option ring at a density of 4 x ¹⁰ cells/well, and cultured at 37°C and 5% _CO2 for 2 days to allow the melanocytes to attach to the vidrigel membrane on the outer surface of the culture insert.

Next, the culture medium was removed from the wells, the option ring was removed, and the culture inserts with melanocytes attached to the vidrigel membrane on the outer surface (lower side) were inserted into the wells of a 12-well plate containing HUMEDIA-KG2 medium (Kurabo Industries, Ltd.) and fixed. Human normal keratinocytes (NHEK(NB), Kurabo Industries, Ltd.) suspended in HUMEDIA-KG2 medium were then seeded into the culture inserts at 1 x ¹⁰ cells/well and cultured at 37°C in 5% _CO2 for 7 days.

Next, the culture inserts were removed from the wells 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.

[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.

[Comparative Example 2]
Human melanoma cells (HMV-II, RCB368) were cultured in HAMF12 medium containing 10% FBS (fetal bovine serum) in a 75 ^cm flask at 37°C and 5% _CO2 until confluent. After culture, 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 (1,000 × g, 5 minutes, 4°C). The resulting supernatant was transferred to a microtube and centrifuged (1,000 × g, 5 minutes, 4°C). The supernatant was transferred to a new microtube and centrifuged (20,000 × g, 5 minutes, 4°C). The supernatant was then removed. A melanosome solution was prepared by adding 0.05 mL of PBS(-) to the resulting pellet.

HUMEDIA-KG2 medium (Kurabo Industries, Ltd.) was added to the wells of a 12-well plate containing Ad-MED Vidrigel 2 (12-well type, manufactured by Kanto Chemical Co., Inc.), and a culture insert was then inserted and fixed. Human normal keratinocytes (NHEK(NB), manufactured by Kurabo Industries, Ltd.) suspended in HUMEDIA-KG2 medium were then seeded into the culture insert at 1 x ¹⁰ cells/well, and 50 μl of the melanosome solution prepared above was added. The culture insert was then cultured at 37°C and 5% _CO for 7 days. Thus, a keratinocyte-attached vidrigel membrane incorporating melanosomes was obtained.

[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.

2. Evaluation of Liquid-Permeable Membranes with Keratinocytes Attached The keratinocyte-attached vidrigel membranes obtained in Example 1 and Comparative Examples 1 to 3 were stained with Fontana-Masson to visualize melanin, and the surface on 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.

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.

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 rapamycin was added to the culture inserts, 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 rapamycin. Rapamycin is a compound that has been suggested to have melanosome-degrading properties.

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

Furthermore, after culturing, the cells were detached from the cell adhesion membrane, and 1 x ¹⁰⁵ cells were immersed in 0.1 ml of 1N aqueous sodium hydroxide solution and disrupted by ultrasound. After thorough stirring, the cells were incubated at 80°C for 20 minutes to extract melanin, and the absorbance at 450 nm was measured to determine the amount of melanin in the keratinocytes.

Figure 3 shows the results of Fontana-Masson staining and optical microscopic observation, and Table 1 shows the results of measuring the amount of melanin in the keratinocytes. As a result, when cultured with the addition of rapamycin, a decrease in melanin amount was observed compared to the control, confirming that the keratinocyte-attached vidrigel membrane prepared in Example 1 can be used to correctly evaluate melanosome degradation activity.

Claims (5)

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;
The evaluation method includes 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 melanosomes in the keratinocytes of the evaluation model; and a fourth step of determining the effect of the test substance on promoting the degradation of melanosomes based on the amount of melanin or melanosomes measured in the third step. The evaluation method according to claim 1, wherein in the first step, the keratinocytes and melanocytes are co-cultured at a distance by dividing the culture vessel using a culture insert having a liquid-permeable membrane at the bottom, thereby allowing the keratinocytes to phagocytose melanosomes. 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;
A screening method comprising: step III of incubating the evaluation model obtained in step II in the presence of a candidate substance, and then measuring the amount of melanin or melanosomes in the keratinocytes of the evaluation model; and step IV of selecting a candidate substance that reduces the amount of melanin or melanosomes in step III as one that has the effect of promoting melanosome degradation. The screening method according to claim 3, wherein in step I, the keratinocytes and melanocytes are co-cultured at a distance by dividing the culture vessel using a culture insert having a liquid-permeable membrane at the bottom, thereby allowing the keratinocytes to phagocytose the melanosomes. Use of an evaluation model for evaluating the melanosome degradation promoting effect of a test substance ,
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 .