Method for evaluating the effects of light irradiation on melanin production in melanocytes

Abstract

This invention provides a method for evaluating the effects of light irradiation on melanin production in melanocytes. [Solution] A method for evaluating the effect of light irradiation on melanin production by melanocytes, comprising irradiating melanocytes with light, culturing the light-irradiated melanocytes in a culture medium, and quantifying the amount of melanin produced by the melanocytes.

Description

[Technical Field]

[0001] This disclosure relates to a method for evaluating the effect of light irradiation on melanin production in melanocytes, a method for determining light irradiation conditions to suppress melanin production in melanocytes, a method for determining light irradiation conditions to promote melanin production in melanocytes, a skin treatment method for suppressing melanin production in the skin, a skin treatment method for promoting melanin production in the skin, and a cosmetic device. [Background technology]

[0002] Melanin, produced by melanocytes in the epidermis, is the cause of skin pigmentation. Beauty devices and other devices have been developed to reduce melanin production by melanocytes through light irradiation (Patent Document 1). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2024 / 101301 brochure [Overview of the project] [Problems that the invention aims to solve]

[0004] However, no suitable method is known to evaluate the effect of light irradiation on melanin production in melanocytes. The object of this disclosure is to provide a method for evaluating the effect of light irradiation on melanin production in melanocytes. [Means for solving the problem]

[0005] This disclosure includes the following aspects: [1] Light irradiation of melanocytes, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, A method for evaluating the effects of light irradiation on melanin production in melanocytes, including [specific example]. [2] Light irradiation of melanocytes, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, When the amount of melanin produced by melanocytes decreases due to light irradiation, the conditions of that light irradiation are selected as light irradiation conditions to suppress melanin production by melanocytes. A method for determining light irradiation conditions to suppress melanin production in melanocytes, including the following. [3] Light irradiation of melanocytes, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, When the amount of melanin produced by melanocytes increases due to light irradiation, the conditions of that light irradiation are selected as light irradiation conditions to promote melanin production by melanocytes. A method for determining light irradiation conditions to promote melanin production in melanocytes, including the conditions mentioned above. [Effects of the Invention]

[0006] This disclosure provides a method for evaluating the effect of light irradiation on melanin production in melanocytes. [Brief explanation of the drawing]

[0007] [Figure 1] This figure shows the preferred FBS concentration (0%~10%(v / v)) in the DMEM for melanin production induction when the cell seeding density is 0.8 × 10⁴ cells / cm² (7 × 10⁴ cells / well, 6-well plate) and there are three light irradiations (505 nm, illuminance: 12 mW / cm², irradiation time per irradiation: 10 minutes), and the preferred melanocyte seeding density when the light irradiation is three times (505 nm, illuminance: 12 mW / cm², irradiation time per irradiation: 10 minutes) and there is a preferred FBS concentration of 2%(v / v) in the DMEM for melanin production induction (n=2 or 3). [Figure 2] This graph shows the percentage of melanin production by melanocytes (compared to 100%) under various light irradiation conditions, with the control group set to 100%, at a cell seeding density of 0.8 × 10⁴ cells / cm² and FBS 2% (v / v). A shows the schedule of the evaluation system, B shows the percentage of melanin production (compared to 100%) when light irradiation (505nm) was performed at an illuminance of 12 mW / cm² for 7.5 minutes once or once for 10 minutes (n=4), and C shows the percentage of melanin production (compared to 100%) when light irradiation (505nm) was performed at an illuminance of 12 mW / cm² for 3 minutes three times, 5 minutes three times, 7.5 minutes three times, or 10 minutes three times (n=4). Each graph is shown with mean + / - standard deviation. [Figure 3] The graphs show the intracellular melanin content, melanocyte melanin production, and melanin / protein ratio (%) under various light irradiation conditions (505 nm), with the control group set to 100%, under a cell seeding density of 0.8 × 10⁴ cells / cm² (7 × 10⁴ cells / well, 6-well plate) and an FBS concentration of 2% (v / v) in DMEM for melanin production induction (n=4). Each graph is shown with mean + / - standard deviation. [Figure 4] The graphs show the intracellular melanin content, melanocyte melanin production, and melanin / protein ratio (%) under various light irradiation conditions (505 nm), with the control group set to 100%, under a cell seeding density of 1.5 × 10⁴ cells / cm² (14 × 10⁴ cells / well, 6-well plate) and an FBS concentration of 4% (v / v) in DMEM for melanin production induction (n=3). Each graph is shown with mean + / - standard deviation. [Figure 5] This graph shows the evaluation system schedule (A) when the culture period before melanocyte harvesting is extended, and the percentage of melanin production by melanocytes compared to the control group (100%). Each graph is shown with mean + / - standard deviation (n=2). [Modes for carrying out the invention]

[0008] The following describes in detail one embodiment of the present disclosure, but the scope of the present disclosure is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the present disclosure. Each embodiment disclosed herein can be combined with any other features disclosed herein. Each configuration and combination thereof in each embodiment is an example, and additions, omissions, substitutions, and other modifications can be made as appropriate without departing from the spirit of the present disclosure. If multiple upper and lower limits are given for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range. The lower and / or upper limits of the numerical ranges described herein may be replaced with numerical values ​​within that range, as shown in the examples. The expression "X~Y" indicating a numerical range means "X or greater and Y or less". If a particular description given for one embodiment also applies to other embodiments, that description may be omitted in the other embodiments. To avoid limiting the application of the doctrine of equivalents to the claims, each numerical measure can be interpreted by taking into account at least the number of significant figures reported and by applying the usual rounding. Any numerical range or value may essentially include the range of errors that inevitably arise from the standard deviation found in each of those test measurements. Unless otherwise specified in the embodiments and examples, use the methods described in standard protocol collections such as J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or modified or altered methods thereof. Furthermore, when using commercially available reagent kits or measuring instruments, use the protocols provided with them unless otherwise specified.

[0009] [First Embodiment (Evaluation Method)] <Method for evaluating the effect of light irradiation on melanin production in melanocytes> The evaluation method according to this embodiment is: Irradiating melanocytes with light, The process of culturing light-irradiated melanocytes in a culture medium. To quantify the amount of melanin produced by melanocytes, This method includes evaluating the effects of light irradiation on melanin production in melanocytes.

[0010] In one embodiment, the effect of light irradiation on melanin production in melanocytes is an increase or decrease in the amount of melanin produced by melanocytes due to light irradiation.

[0011] [(a) Irradiating melanocytes with light] (Melanocytes) Melanocytes are cells that produce melanin, and are also called melanocytes or melanin-producing cells. They are mainly found in the basal layer of the epidermis of the skin of animals such as humans.

[0012] Melanocytes are not limited by their origin animal, as long as they contain melanocytes. The animal may be a mammal, or any other animal such as a bird, reptile, amphibian, or fish, but it is preferable that it be a mammal. The mammal may be a human or a non-human animal, but it is preferable that it be a human. Non-human animal species may be, for example, monkeys, dogs, cats, horses, cattle, pigs, sheep, goats, rabbits, guinea pigs, hamsters, mice, and / or rats, and are not limited by their intended use, such as livestock, pets, or laboratory animals.

[0013] In one embodiment, the melanocytes are human or mouse-derived melanocytes.

[0014] Melanocytes are not limited by the developmental stage or age of the animal from which they originate, and may be of any age from fetus or neonatal to adult.

[0015] Melanocytes are not limited by the type of animal tissue from which they originate, as long as the tissue contains melanocytes, and may originate from, for example, skin (including, for example, the face, neck, body, lower limbs, and external genitalia), eyes (including the retina and choroid), or mucous membranes (including the oral cavity, esophagus, and intestines), but are not limited to these.

[0016] Melanocytes may be cells present in the tissues of an animal, cells present in tissues isolated from an animal, or cells isolated from an animal, or primary cultured cells, subcultured cells, or established cell lines isolated from an animal, or cells produced by differentiating stem cells under conditions well known to those skilled in the art.

[0017] If melanocytes are cultured cells, primary cultures, subcultures, or established human or mouse melanocyte lines available from reagent companies, cell banks, etc., can also be used.

[0018] Melanocytes may be normal cells or cancerous cells (e.g., malignant melanoma cells). Normal or cancerous cells may have gene mutations related to or unrelated to melanin production. When melanocytes are melanoma cells, examples include mouse-derived melanoma cell lines (B16) (including B16 4A5) and human-derived melanoma cell lines (HMV-II, MNT-1, etc.). Cell-formed melanocytes and melanoma cells can be obtained from reagent companies and research institutions (such as RIKEN RBC).

[0019] If melanocytes are cells present in the tissue of an animal, or cells present in tissue isolated from an animal, then light irradiation is performed on the cells within the tissue. In this case, light may be irradiated from any side of the animal tissue as long as the light can reach the melanocytes. For example, if the tissue is skin, light may be irradiated onto the skin from the outside of the animal's body, thereby irradiating the melanocytes present in the epidermis of the skin.

[0020] When melanocytes are cells isolated from an animal, primary cultured cells thereof, subcultured cells, or established cell lines, and light irradiation is performed in vitro, light irradiation may be performed on melanocytes under culture conditions well known to those skilled in the art, or, in the case of culture in liquid medium, the liquid medium may be temporarily removed at the time of light irradiation, and light irradiation may be performed on melanocytes attached to the culture substrate (plate, dish, tube, etc.).

[0021] When light irradiation is performed in vitro, the culture medium and / or its acidity used for culturing melanocytes before and / or during light irradiation, the culture temperature, CO2 environment, etc., may be as described in "(b)" below. In one embodiment, it is preferable to culture melanocytes before and / or during light irradiation in a medium containing a higher concentration of serum than that used in culture "(b)". For example, the serum concentration is 2% to 15% (v / v), more preferably 5% to 12% (v / v), even more preferably 8% to 10% (v / v), and even more preferably 10% (v / v). Examples of serum types and preferred examples are described in "(b)" below.

[0022] In one embodiment, melanocyte culture before and / or during light irradiation may be carried out in a liquid medium of DMEM (Dulbeccoo's modified Eagle medium) containing 8-10% (v / v) fetal bovine serum at 36-37°C and in a 3-10% CO2 environment.

[0023] (Light irradiation) The wavelength of light irradiation can be appropriately selected by a person skilled in the art from the wavelengths at which the effects of light irradiation on melanocytes are to be evaluated. The light is preferably visible light. The visible light may be green wavelength or yellow wavelength. Here, the green wavelength may be 490 nm to 570 nm, and in one embodiment, it may be 490 nm to 525 nm, 495 nm to 525 nm, or 505 nm. The yellow wavelength may be 570 nm to 590 nm.

[0024] The light may have a peak wavelength range of 490 nm to 570 nm. In one embodiment, the peak wavelength range may be 490 nm to 525 nm, 495 nm to 525 nm, or 505 nm. That is, the light may be a plurality of light sources having a peak wavelength range of 490 nm to 570 nm and having different wavelengths within and / or outside the peak wavelength range.

[0025] Because the light is visible light, the evaluation method according to this embodiment makes it easier to evaluate the effect of light irradiation on melanin production in melanocytes with higher sensitivity.

[0026] In one embodiment, multiple different wavelengths of light can be irradiated simultaneously, alternately, and / or sequentially, and the effect of such combinations of light irradiation on melanin production in melanocytes can be evaluated.

[0027] Light is not limited by its light source; for example, it may be natural light, light emitted from a lighting fixture, or a light source provided in a light irradiation device. In the case of a light irradiation device, it may be, for example, a fluorescent lamp, LED (light-emitting diode), laser light, IPL (intense pulsed light), etc.

[0028] In one embodiment, light irradiation is performed by accumulating irradiation dose (mJ / cm²). 2 ) may be 1000 to 200000, 3000 to 200000, 3000 or more and less than 180000, 3000 to 150000, 4000 to 100000, 5000 to 90000, or 54000 to 90000, and may be any range with one of these as the lower limit and one of the other as the upper limit, but is not limited to these. Cumulative irradiation dose (mJ / cm²) 2 A value between 3000 and 200000 allows for more sensitive evaluation of the effects of light irradiation on melanin production in melanocytes. Furthermore, the cumulative irradiation dose (mJ / cm²) 2 When the value is less than 180,000, more preferably 90,000 or less, and even more preferably less than 90,000, toxicity to melanocytes is less likely to occur, and therefore it becomes easier to evaluate the effect of light irradiation on melanin production in melanocytes within a range that is not affected by toxicity. In one embodiment, the cumulative irradiation dose (mJ / cm²) 2The values ​​of ) are 1,000 or more and less than 18,000, more preferably 3,000 or more and less than 180,000, even more preferably 3,000 or more and less than 90,000, even more preferably 3,000 or more and less than 90,000, even more preferably 4,000 or more and less than 90,000, even more preferably 5,000 or more and less than 90,000, and even more preferably 54,000 or more and less than 90,000, which makes it easier to evaluate the effect of light irradiation on melanin production in melanocytes with higher sensitivity, and also makes it easier to evaluate the effect of light irradiation on melanin production in melanocytes within a range that is not affected by toxicity.

[0029] Here, the cumulative irradiation dose (mJ / cm²) 2 ) is illuminance (mW / cm 2 It can be calculated using the following formula based on the irradiation time (seconds) and the number of irradiations. [Formula] Cumulative irradiation dose (mJ / cm²) 2 ) = Illuminance × Irradiation time × Number of irradiations

[0030] Cumulative irradiation dose (mJ / cm²) 2 If the value is between 3,000 and 200,000, the illuminance, irradiation time, and number of irradiations can be appropriately adjusted by a person skilled in the art so that the cumulative irradiation amount falls within the above range.

[0031] The irradiation time, number of irradiations, and illuminance of light irradiation can be appropriately selected by a person skilled in the art from the irradiation time, number of irradiations, and illuminance for which the effect of light irradiation on melanocytes is to be evaluated. For example, the number of irradiation sessions may be 1 to 100, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sessions. Any range may be used with one of these as the lower limit and one of the other as the upper limit, but is not limited to these. If light irradiation is performed multiple times, the interval between each light irradiation may be, for example, 30 minutes to 3 days, or 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 50 hours, 70 hours, or 72 hours (3 days), and may be any range with one of these as the lower limit and one of the other as the upper limit, but is not limited to these. For example, the irradiation time per irradiation may be 30 seconds to 60 minutes (3,600 seconds), may be 30 seconds, 1 minute, 3 minutes, 5 minutes, 7.5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes, and may be any range with any one of these as the lower limit and any other one as the upper limit, but is not limited thereto. For example, the illuminance (mW / cm 2 ) may be 1 to 500, may be 1 to 400, may be 1 to 300, may be 1 to 200, may be 3, 3.5, 3.7, 5, 6, 6.5, 10, 12, 15, 18, 20, 21, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200, and may be any range with any one of these as the lower limit and any other one as the upper limit, but is not limited thereto.

[0032] When light irradiation is performed multiple times, the irradiation time and / or illuminance of each light irradiation may be different in each light irradiation.

[0033] In one embodiment, the light irradiation may be any of the following combinations of illuminance, irradiation time, and number of irradiations at a wavelength of 505 nm. · Illuminance: 12 mW / cm 2 , irradiation time: 7.5 minutes, number of irradiations: 1 time · Illuminance: 12 mW / cm 2 , irradiation time: 10 minutes, number of irradiations: 1 time · Illuminance: 100 mW / cm 2 , irradiation time: 3 minutes, number of irradiations: 1 time · Illuminance: 12 mW / cm 2 , irradiation time: 5 minutes, number of irradiations: 1 time · Illuminance: 6.5 mW / cm 2 , irradiation time: 10 minutes, number of irradiations: 3 times · Illuminance: 12 mW / cm 2、 Irradiation time: 5 minutes, number of irradiations: 3 times · Illuminance: 12 mW / cm 2 , irradiation time: 7.5 minutes, number of irradiations: 3 times · Illuminance: 12 mW / cm 2 , irradiation time: 10 minutes, number of irradiations: 3 times ·Illuminance: 18mW / cm 2 Irradiation time: 5 minutes, Number of irradiations: 3 times ·Illuminance: 18mW / cm 2 Irradiation time: 10 minutes, Number of irradiations: 3 ·Illuminance: 100mW / cm 2 Irradiation time: 3 minutes, Number of irradiations: 3 times ·Illuminance: 100mW / cm 2 Irradiation time: 5 minutes, Number of irradiations: 3 times ·Illuminance: 12mW / cm 2 Irradiation time: 20 minutes, Number of irradiations: 3

[0034] In one embodiment, if the melanocytes are cells present in the tissue of an animal, or cells present in tissue isolated from an animal, the method may further include a step of irradiating the melanocytes with light and then isolating the melanocytes from the tissue. The isolation of melanocytes from tissue can be carried out by methods well known to those skilled in the art. For example, by finely fragmenting tissue with a blade or similar tool and treating it multiple times with a proteolytic enzyme solution, the epidermis can be separated and epidermal cells dispersed. Subsequently, cell seeding can be performed, and during the first passage, a cell dispersion can be obtained using a proteolytic enzyme solution, and then melanocytes can be isolated using a proteolytic enzyme solution.

[0035] (b) Culturing light-irradiated melanocytes in a culture medium. (culture medium) The culture medium is not limited to a range capable of culturing melanocytes, and can be appropriately selected from cell culture media well known to those skilled in the art. In one embodiment, the conditions for the culture medium described below may be maintained under the same conditions continuously from the start to the end of the culture, or the conditions may be adjusted as needed during a certain period of the culture.

[0036] Non-specific examples of culture media include DMEM (Dulbecco's Modified Eagle Medium), RPMI1640 medium, DMEM / F12 medium, MEM medium, Ham F-10 medium, Ham F-12 medium, and 199 medium (M199).

[0037] The acidity of the culture medium can be set appropriately considering the animal from which the melanocytes originate, but for example, it may be pH 6.0 to 8.0 or pH 6.8 to 7.5, and in the case of mammalian origin, pH 7.0 to 7.4 is preferred.

[0038] The culture medium is not limited by its form; for example, it may be a liquid medium or a solid medium. In the case of solid culture media, the medium may be prepared in a solid form using agar, gelatin, or the like. Considering the treatment during and after cell culture, a liquid culture medium is preferable.

[0039] In one embodiment, the culture medium preferably contains serum. If the culture medium contains serum, the concentration is preferably greater than 0.2% and less than 5% (v / v), more preferably between 0.5% and 4.5% (v / v), and even more preferably between 1% and 4% (v / v). If it is between 1% and 4% (v / v), it may be 1%, 2%, 3%, or 4%, and may be any range with one of these as the lower limit and one of the others as the upper limit, but is not limited to these. Including serum in the culture medium at a concentration greater than 0.2% but less than 5% (v / v) makes it easier to evaluate the effects of light irradiation on melanin production in melanocytes with higher sensitivity.

[0040] The serum can be appropriately selected from serums that are well known to those skilled in the art and can be used as culture media for cell culture, and is not limited by the animal from which the serum originates. Non-limiting examples of animals from which serum originates include mammals such as cattle, horses, sheep, goats, pigs, llamas, dogs, donkeys, cats, rabbits, guinea pigs, hamsters, rats, mice, and humans, as well as birds such as chickens. The source animal for the serum is not limited by its developmental stage or age. For example, it may be a fetus, a newborn, a young individual, or an adult. In one embodiment, the serum is fetal bovine serum (FBS, FCS).

[0041] In one embodiment, the culture medium contains FBS at a concentration of more than 0.2% and less than 5% (v / v), preferably at a concentration of 0.5 to 4.5% (v / v), and more preferably at a concentration of 1 to 4% (v / v). In one embodiment, the culture medium is a liquid culture medium containing FBS at a concentration of more than 0.2% and less than 5% (v / v), preferably at a concentration of 0.5 to 4.5% (v / v), and more preferably at a concentration of 1 to 4% (v / v).

[0042] In one embodiment, the culture medium may contain one or more components that can promote and / or induce melanin production by melanocytes. Examples of such components include melanocyte-stimulating hormone, pituitary hormones, female hormones, and xanthine derivatives. Examples of melanocyte-stimulating hormones (MSH, melanotropin) include α-MSH, β-MSH, or γ-MSH. Examples of pituitary-secreted hormones include ACTH (adrenocorticotropic hormone), LH (luteinizing hormone), or FSH (follicle-stimulating hormone). Examples of female hormones include estradiol, estriol, or progesterone. Examples of xanthine derivatives include theophylline or caffeine.

[0043] In one embodiment, the culture medium may contain α-MSH and / or theophylline.

[0044] The concentrations of the above components in the culture medium can be appropriately set by those skilled in the art. For example, in the case of melanocyte-stimulating hormone, pituitary hormones, or female hormones, the concentrations may be 1 nM to 1000 nM (1 μM), 1 nM to 500 nM, 10 nM to 500 nM, or 50 nM to 200 nM, respectively. However, 80 nM to 120 nM is preferred, 90 nM to 110 nM is more preferred, and 100 nM is even more preferred. For example, in the case of xanthine derivatives, the respective concentrations may be 1 μM to 1000 μM (1 mM), 1 μM to 500 μM, 10 μM to 500 μM, or 50 μM to 200 μM, but 80 μM to 120 μM is preferred, 90 μM to 110 μM is more preferred, and 100 μM is even more preferred.

[0045] The inclusion of the above components in the culture medium makes it easier to increase melanin production by melanocytes, and to evaluate the effect of light irradiation on melanin production by melanocytes with greater sensitivity.

[0046] In one embodiment, the culture medium preferably contains α-MSH at a concentration of 80 nM to 120 nM, more preferably 90 nM to 110 nM, and even more preferably 100 nM, and / or in one embodiment, the culture medium preferably contains theophylline at a concentration of 80 μM to 120 μM, more preferably 90 μM to 110 μM, and even more preferably 100 μM (0.1 mM).

[0047] The culture medium may contain additives commonly added to culture media in cell culture, not just for melanocytes. Non-limiting examples of additives include amino acids (including L-glutamine), sodium bicarbonate, and antibiotics. In one embodiment, the culture medium contains 1 to 10 mM, preferably 1 to 5 mM, and more preferably 2 mM of L-glutamine.

[0048] (culture) The culture conditions for melanocytes are not limited to the range in which melanocytes can be cultured, and can be appropriately set by those skilled in the art, taking into consideration the type of melanocyte, the type of culture medium, etc.

[0049] In one embodiment, the culture temperature can be appropriately set by those skilled in the art, taking into consideration the animal species from which the melanocytes originate. However, when the melanocytes are derived from mammals, the culture temperature is preferably 20°C to 50°C, more preferably 25°C to 45°C, even more preferably 30°C to 40°C, and even more preferably 36°C to 37°C. In one embodiment, the culture temperature is 37°C.

[0050] In one embodiment, the atmospheric environment for culture can be appropriately set considering the culture medium conditions and / or the animal species from which the melanocytes originate, but it may also be carried out under a CO2 concentration control environment, for example, under a 1-15% CO2 environment, preferably under a 3-10% CO2 environment, more preferably under a 4-7% CO2 environment, and even more preferably under a 5% CO2 environment.

[0051] The culture should be carried out for a sufficient amount of time for melanocytes to produce melanin, but in one embodiment, the culture time is 1 to 10 days, preferably 2 to 5 days, more preferably 3 to 4 days, and even more preferably 3 days from the first light irradiation, or 24 to 240 hours, preferably 48 to 120 hours, more preferably 72 to 96 hours, and even more preferably 72 hours from the first light irradiation.

[0052] In one embodiment, if light irradiation is performed multiple times (see "(a)" "light irradiation"), the process of irradiating melanocytes with light and culturing the light-irradiated melanocytes in a culture medium may be repeated alternately multiple times.

[0053] The seeding density of melanocytes is, for example, 0.05 × 10⁻⁶. 4 cells / cm 2 ~6.0×10 4 cells / cm 2 It may also be 0.1 × 10 4 cells / cm 2 Super and 2.2 x 104 cells / cm 2 Less than 0.3 × 10 is preferable, 4 cells / cm 2 ~2.0×10 4 cells / cm 2 More preferably, 0.4 × 10 4 cells / cm 2 ~1.5×10 4 cells / cm 2 That is even more preferable. 0.4 × 10 4 cells / cm 2 ~1.5×10 4 cells / cm 2 In that case, 0.4 × 10 4 cells / cm 2 , 0.5 × 10 4 cells / cm 2 , 0.6 × 10 4 cells / cm 2 , 0.7 × 10 4 cells / cm 2 , 0.8 × 10 4 cells / cm 2 , 0.9 × 10 4 cells / cm 2 , 1.0 × 10 4 cells / cm 2 , or 1.5 × 10 4 cells / cm 2 It may be any range with one of these as the lower limit and one of the other as the upper limit, but it is not limited to these. Melanocyte seeding density is 0.1 × 10 4 cells / cm 2 Super and 2.2 x 10 4 cells / cm 2 When the value is less than this, the effect of light irradiation on melanin production in melanocytes can be evaluated with higher sensitivity.

[0054] Each of the above components for culturing light-irradiated melanocytes may be combined in any combination of numerical ranges. In one embodiment, light-irradiated melanocytes are 0.1 × 104 cells / cm 2 Super and 2.2 x 10 4 cells / cm 2 At a seeding density of less than 0.1 × 10⁻¹⁶, the cells may be cultured for 3 days after light irradiation in a liquid medium containing 1-4% (v / v) FBS, 80 nM-120 nM α-MSH, and 80 μM-120 μM theophylline. In one embodiment, the light-irradiated melanocytes were 0.1 × 10⁻¹⁶ 4 cells / cm 2 Super and 2.2 x 10 4 cells / cm 2 Culture may be performed at a seeding density of less than 100%, in a liquid medium containing 1-4% (v / v) FBS, 80 nM-120 nM α-MSH, and 80 μM-120 μM theophylline, with the first light irradiation followed by second and third light irradiations every 24 hours, and cultured for 3 days from the first light irradiation.

[0055] [(c) Quantifying the amount of melanin produced by melanocytes] The amount of melanin produced by melanocytes can be quantified by methods well known to those skilled in the art. For example, melanocytes can be harvested after culture, dissolved in a 1 mol / l aqueous sodium hydroxide solution containing 10 wt% dimethyl sulfoxide (DMSO), and then the amount of melanin produced can be measured from the absorbance at 405 nm. Alternatively, by measuring melanocyte-derived proteins using a protein quantification method well known to those skilled in the art, the amount of melanin per unit amount of melanocyte-derived protein (melanin production amount / cell-derived protein amount) may be calculated and used as the melanin production amount of melanocytes. Proteins can also be quantified using commercially available kits, such as the Pierce® BCA Protein Assay Kit (catalog number: 23225, manufactured by Thermo Scientific) or the RC DCTM Protein Assay Kit (manufactured by BioRad). Here, the amount of cell-derived protein reflects the number of living cells (number of living melanoma cells). For example, if the amount of cell-derived protein is lower in the light-irradiated group compared to the control group, it can be said that the light irradiation is toxic to the cells. Therefore, in one embodiment, if the amount of melanin per unit amount of melanocyte-derived protein (melanin production amount / cell-derived protein amount) is used as the amount of melanin produced by melanocytes, it is possible to determine whether or not the light irradiation is toxic.

[0056] Based on the quantified amount of melanin, the effect of light irradiation on melanin production in melanocytes can be evaluated. More specifically, for example, it is possible to evaluate whether light irradiation increased or decreased melanin production in melanocytes. The evaluation can be determined, for example, by comparing with a control group that did not receive light irradiation, or by comparing with a pre-established reference value or reference range. The comparison with the control group, reference value, or reference range may be performed, for example, by any statistical method.

[0057] The "effect of light irradiation" that is the subject of evaluation in the evaluation method according to this embodiment may be the effect of a specific light wavelength, integrated irradiation dose, illuminance, irradiation time, and / or number of irradiations. Based on this evaluation, it may be used to determine desired light irradiation conditions (e.g., light wavelength, integrated irradiation dose, illuminance, irradiation time, and / or number of irradiations) as follows.

[0058] The following describes a method for evaluating the effect of light irradiation on melanin production in melanocytes according to one embodiment, following the procedure of one embodiment.

[0059] (1) Place B16 mouse melanoma cells (B16 4A5) in a 6-well plate in a 0.4 × 10⁻¹⁶ well. 4 cells / cm 2 ~1.6×10 4 cells / cm 2 Seeds are seeded at a density of 10% (v / v) FBS and cultured in DMEM at 37°C under 5% CO2 conditions. (2) 24 hours after the start of culture, replace with DMEM (DMEM for melanin production induction) containing 1-4% (v / v) FBS, 100 nM α-MSH, and 100 μM theophylline. (3) After culturing for 1 to 6 hours, perform the first light irradiation. (4) After the first light irradiation, culture under the same conditions for 3 days. If more than one irradiation is performed, it should be done every 24 hours after the first light irradiation. (5) Wash the melanoma cells with PBS(-), collect them, and dissolve them in a 1 mol / l sodium hydroxide aqueous solution containing 10 wt% DMSO. Measure the amount of melanin produced by the melanoma cells based on the absorbance at 405 nm. (6) Use a protein assay kit to measure melanoma cell-derived proteins. (7) Calculate the amount of melanin per unit of melanoma cell-derived protein, and use this as the amount of melanin produced by melanocytes (melanoma cells). (8) The effect of light irradiation on melanin production in melanocytes will be evaluated by comparing it with the control group (no light irradiation).

[0060] <Method for determining light irradiation conditions to suppress melanin production in melanocytes> In one embodiment, the "method for evaluating the effect of light irradiation on melanin production in melanocytes" can be used in a method for determining light irradiation conditions to suppress melanin production in melanocytes, which includes the said method.

[0061] In other words, "a method for determining light irradiation conditions to suppress melanin production in melanocytes" is, Irradiating melanocytes with light, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, This includes selecting the light irradiation conditions as light irradiation conditions for suppressing melanin production by melanocytes when the amount of melanin produced by melanocytes decreases due to light irradiation.

[0062] Examples and preferred examples of irradiating melanocytes with light, culturing the irradiated melanocytes in a culture medium, and quantifying the amount of melanin produced by the melanocytes are as described in (a) to (c) above.

[0063] [(d-1) When the amount of melanin produced by melanocytes decreases due to light irradiation, select the conditions for said light irradiation as conditions for suppressing melanin production by melanocytes.] Whether or not melanin levels decreased due to light irradiation can be determined, for example, by comparing the results with a control group that did not receive light irradiation, or by comparing them with a pre-established reference value or reference range. The comparison with the control group, reference value, or reference range may be performed, for example, by any statistical method.

[0064] If the amount of melanin produced by melanocytes is reduced, the light irradiation conditions can be selected as light irradiation conditions to suppress melanocyte production.

[0065] The light irradiation conditions selected in this manner can be applied, for example, to a skin treatment method described in the second embodiment or a beauty device described in the third embodiment, and can be used to prevent or reduce melanin deposition in the skin, etc.

[0066] <Method for determining light irradiation conditions to promote melanin production in melanocytes> In one embodiment, the "method for evaluating the effect of light irradiation on melanin production in melanocytes" can be used in a method for determining light irradiation conditions to promote melanin production in melanocytes, which includes the said method.

[0067] In other words, "a method for determining light irradiation conditions to promote melanin production in melanocytes" is, Irradiating melanocytes with light, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, This includes selecting the conditions for light irradiation as conditions for promoting melanin production by melanocytes when the amount of melanin produced by melanocytes increases due to light irradiation.

[0068] Examples and preferred examples of irradiating melanocytes with light, culturing the irradiated melanocytes in a culture medium, and quantifying the amount of melanin produced by the melanocytes are as described in (a) to (c) above.

[0069] [(d-2) If the amount of melanin produced by melanocytes increases due to light irradiation, select the conditions for said light irradiation as conditions for promoting melanin production by melanocytes.] Whether or not melanin production was increased by light irradiation can be determined, for example, by comparing the results with a control group that did not receive light irradiation, or by comparing them with a pre-established reference value or reference range. The comparison with the control group, reference value, or reference range may be performed, for example, by any statistical method.

[0070] If the amount of melanin produced by melanocytes is increasing, the light irradiation conditions can be selected as light irradiation conditions to promote melanocyte production.

[0071] The light irradiation conditions selected in this manner can be applied, for example, to the skin treatment method described in the second embodiment or the beauty device described in the third embodiment, and can be used to promote melanin production and / or melanin deposition in human hair, including scalp hair (prevention of gray hair or darkening of hair).

[0072] [Second Embodiment (Skin Treatment Method)] The skin treatment method according to this embodiment is a skin treatment method for suppressing melanin production in the skin, which includes irradiating the skin with light according to the light irradiation conditions determined by the "Method for determining light irradiation conditions to suppress melanin production in melanocytes" described in the first embodiment. In one embodiment, the skin treatment method can be used to prevent or reduce melanin deposition in the skin. Another skin treatment method according to this embodiment is a skin treatment method for promoting melanin production in the skin, which includes irradiating the skin with light according to the light irradiation conditions determined by the "Method for determining light irradiation conditions to promote melanin production in melanocytes" described in the first embodiment. In one embodiment, this skin treatment method can be used to promote melanin production and / or melanin deposition (prevention of gray hair or darkening of hair) in hair, including human scalp hair.

[0073] In any of the above skin treatment methods, medical procedures may be excluded.

[0074] Skin treatment is preferably performed on the skin of a living organism. The target animal is not limited to the range of animals having melanocytes, and the animal may be, for example, a mammal or any other animal such as a bird, reptile, amphibian, or fish, but it is preferable that it be a mammal. The mammal may be a human or a non-human animal, but it is preferable that it be a human. Non-human animal species may be, for example, a monkey, dog, cat, horse, cow, pig, sheep, goat, rabbit, guinea pig, hamster, mouse, and / or rat, and is not limited by use such as livestock, pet, or laboratory animal.

[0075] In one embodiment, the skin is human skin.

[0076] The method of light irradiation to the skin is not limited, but can be carried out, for example, by using a device having a light irradiation means capable of irradiating under predetermined conditions. Non-limiting examples of the device include medical devices or cosmetic devices described in the third embodiment.

[0077] [Third Embodiment (Medical Device or Cosmetic Device)] The medical or cosmetic device according to this embodiment is a device that includes light irradiation means for irradiating with light according to the light irradiation conditions determined by the "Method for determining light irradiation conditions for suppressing melanin production of melanocytes" described in the first embodiment. In one embodiment, the device is a device that includes light irradiation means for irradiating with light according to one or more of the various conditions described in "light irradiation" of the first embodiment. In one embodiment, the device is a medical or cosmetic device for suppressing melanin production of melanocytes and can be used, for example, to prevent or reduce melanin deposition in the skin. Another medical or cosmetic device according to this embodiment is a device that includes a light irradiation means for irradiating light according to the light irradiation conditions determined by the "method for determining light irradiation conditions to promote melanin production in melanocytes" described in the first embodiment. In one embodiment, the device is a medical or cosmetic device for promoting melanin production in melanocytes and can be used, for example, to promote melanin production and / or melanin deposition in hair, including human scalp hair (prevention of gray hair or darkening of hair).

[0078] The device may, for example, be equipped with a light irradiation means (light source) capable of irradiating light under determined conditions, and have a configuration that allows light from the light source to be irradiated onto the skin. In one embodiment, the light irradiation means may be controlled by a control unit (such as a computer) provided in the device.

[0079] The light irradiation under determined conditions includes illuminance, irradiation time per session, total irradiation time within a certain period (hereinafter, the irradiation time per session and the total irradiation time are collectively referred to as "irradiation time"), number of irradiations, and cumulative irradiation dose. The device may be equipped with means to detect when the appropriate range of light irradiation (for example, at least one of the determined conditions) has been reached by quantitatively measuring or counting the illuminance, irradiation time, and / or number of irradiations used by the user, automatically stop the light irradiation, and / or notify the user that the appropriate range has been reached. The device may also be equipped with means to automatically calculate the cumulative irradiation dose based on the illuminance, irradiation time, and / or number of irradiations used by the user, automatically stop the light irradiation when the light irradiation reaches the appropriate range, and / or notify the user that the appropriate range has been reached. Such notification means are not limited to those that can be notified to the user and may act on any of the senses such as sight, hearing, touch, taste, and smell, but may also include, for example, a display on the surface of the device, a notification sound, or vibration of the device. Having such means makes it possible to provide the user with appropriate light irradiation. In one embodiment, if the appropriate range of other light irradiation conditions differs depending on the light source and / or wavelength used, the device may be equipped with a control unit that enables different appropriate light irradiation for each light source and / or wavelength used. In one embodiment, the device may have a configuration that allows the user to select a desired illuminance from two or more illuminance levels. In that case, the device may include means for counting the selected illuminance, irradiation time, and number of irradiations, sensing when the light irradiation reaches the appropriate range as described above, stopping the light irradiation, and / or notifying the user.

[0080] In one embodiment, the device may include means for registering one or more users. In this case, the device may further include means for understanding the usage status of the device for each registered user over a certain period of time. The certain period may be, for example, one hour, one day, one week, one month, or one year. By understanding the usage status of the device over a certain period of time, the device may include means for sensing when each registered user has reached the appropriate range (determined conditions) of light irradiation during that period, stopping the light irradiation, and / or notifying the user. Having such means makes it possible to irradiate with light within the appropriate range even if the device is used intermittently over a certain period of time. Furthermore, by allowing multiple users to be registered, it becomes possible to irradiate with light within the appropriate range for each user even if the device is used intermittently by multiple users.

[0081] In one embodiment, the device may be portable, with the entire device being graspable by the user's hand, or it may be a stationary type consisting of a fixed device, a movable part, an arm connecting the fixed device and the movable part, etc. In one embodiment, the device may be equipped with various buttons such as an on / off button, a mode switching button, and an intensity adjustment button for the user to control the light irradiation.

[0082] The configuration of equipment other than those described above can be appropriately set by a person skilled in the art.

[0083] A non-limiting list of exemplary embodiments and combinations of exemplary embodiments of this disclosure are disclosed below. [1] Light irradiation of melanocytes, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, A method for evaluating the effects of light irradiation on melanin production in melanocytes, including [specific example]. [2] The method according to [1-1], wherein the culture medium contains more than 0.2% and less than 5% (v / v) serum. [3] Light-irradiated melanocytes in the culture medium 0.1 × 10 4 cells / cm2 Super and 2.2 x 10 4 cells / cm 2 The method according to [1] or [2], wherein the seeds are sown at a density less than [2]. [4] The method according to any one of [1] to [3], wherein the light is visible light. [5] The method according to any one of [2] to [4], wherein the serum is fetal bovine serum. [6] The method according to any of [2] to [4], wherein the serum is at a concentration of 1-4% (v / v). [7] Light irradiation of melanocytes, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, When the amount of melanin produced by melanocytes decreases due to light irradiation, the conditions of that light irradiation are selected as light irradiation conditions to suppress melanin production by melanocytes. A method for determining light irradiation conditions to suppress melanin production in melanocytes, including the following. [8] Light irradiation of melanocytes, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, When the amount of melanin produced by melanocytes increases due to light irradiation, the conditions of that light irradiation are selected as light irradiation conditions to promote melanin production by melanocytes. A method for determining light irradiation conditions to promote melanin production in melanocytes, including the conditions mentioned above. [9] The method according to [7] or [8], wherein the culture medium comprises more than 0.2% and less than 5% (v / v) serum.

[10] Light-irradiated melanocytes in the culture medium 0.1 × 10 4 cells / cm 2 Super and 2.3 x 10 4 cells / cm 2 The method according to any one of [7] to [9], wherein the seeds are sown at a density less than [1].

[11] The method according to any one of [7] to

[10] , wherein the light is visible light.

[12] The method according to any one of [9] to

[11] , wherein the serum is fetal bovine serum.

[13] The method according to any one of [9] to

[12] , wherein the serum is at a concentration of 1-4% (v / v). A skin treatment method (excluding medical procedures) for suppressing melanin production in the skin, comprising irradiating the skin with light under light irradiation conditions determined by the method of any one of

[14] [7] and [9] to

[13] . A skin treatment method (excluding medical procedures) for promoting melanin production in the skin, comprising irradiating the skin with light under light irradiation conditions determined by any one of the methods described in

[15] [8] and [9] to

[13] . A beauty device comprising a light irradiation means for irradiating light according to light irradiation conditions determined by any one of the methods described in

[16] [7] to

[13] . [Examples]

[0084] The present disclosure will be further illustrated by the following examples, but these examples will not limit the interpretation of the present disclosure.

[0085] Example 1: Evaluation system for the effect of light irradiation on melanin production in melanocytes [1. Evaluation Method] 6-hole plate (35mm diameter, 9.6cm) 2 B16 mouse melanoma cells (B16 4A5, obtained from RIKEN RBC) were seeded at the following densities in DMEM (catalog number: 040-30095, Fujifilm Wako Pure Chemical Industries) containing 10% (v / v) FBS (catalog number: 26140-079, Gibco, USA) and 2 mM L-glutamine (catalog number: 073-05391, Fujifilm Wako Pure Chemical Industries). The cells were cultured at 37°C under 5% CO2. (Melanoma cell seeding density) (1) 0.1 × 10 4cells / cm 2 (1×10 4 cells / well, 6-well plate) (2)0.4×10 4 cells / cm 2 (3.5×10 4 cells / well, 6-well plate) (3)0.7×10 4 cells / cm 2 (5×10 4 cells / well, 6-well plate) (4)0.8×10 4 cells / cm 2 (7×10 4 cells / well, 6-well plate) (5)1.5×10 4 cells / cm 2 (14×10 4 cells / well, 6-well plate) (6)2.2×10 4 cells / cm 2 (21×10 4 cells / well, 6-well plate)

[0086] Twenty-four hours after the start of culture, it was replaced with DMEM (DMEM for inducing melanogenesis) containing FBS, 100 nM α-MSH (catalog number: M4135, Sigma-Aldrich, USA), 100 μM theophylline (catalog number: T1633, Sigma-Aldrich, USA), and 2 mM L-glutamine (catalog number: 073-05391, Fujifilm Wako Pure Chemical). After culturing for 2 hours, the first light irradiation at a wavelength of 505 nm was performed under the following conditions. The light source LED used was SMT505 manufactured by Ushio Electric. The FBS in the medium was set to 0, 0.2, 1, 2, 3, 4, 5, or 10% (v / v). The illuminance (output) of the light irradiation was 12 mW / cm 2 as. The light irradiation was performed once or three times. ​​​ After the first light irradiation, melanoma cells were cultured in the same medium at 37°C and 5% CO2 for a further 3 days (72 hours). During this time, in the group that received three light irradiations, light irradiation was performed again 24 hours and 48 hours after the first irradiation, using the same illuminance (power) and irradiation duration as the first irradiation.

[0088] After 3 days of incubation, the cells were incubated for another day under the same conditions.

[0089] In this evaluation method, the control group consisted of those who underwent no light irradiation, but whose other procedures were identical to those of the light-irradiated group.

[0090] (Quantitative determination of melanin production) Cells were washed and collected with PBS(-) (calcium and magnesium-free phosphate-buffered saline) (catalog number: 2810305, MP Biomedicals), and then dissolved in a 1 mol / l sodium hydroxide aqueous solution containing 10 wt% dimethyl sulfoxide (DMSO). Melanin production by melanoma cells was measured based on absorbance at 405 nm. For samples in which melanin production was measured, cell-derived proteins were further measured using the Pierce® BCA Protein Assay Kit (catalog number: 23225, Thermo Scientific). Based on the measurement results, the amount of melanin per unit of melanoma cell-derived protein (melanin production / cell-derived protein) was calculated and defined as the melanin production amount. Unless otherwise specified, the evaluation results below are shown as percentages calculated with the control group set at 100%. Here, the amount of cell-derived protein reflects the number of viable cells (number of viable melanoma cells). For example, if the amount of cell-derived protein is lower in the light-irradiated group compared to the control group, it can be said that the light irradiation is toxic to the cells.

[0091] [2. Evaluation Results] (Recommended fetal bovine serum concentration) For FBS concentrations (0%~10% (v / v)) in DMEM for melanin production induction, the cell seeding density was 0.8 × 10⁻⁶.4 cells / cm 2 (7×10 4 Figure 1 shows the evaluation results for cells / well (6-well plate). This evaluation was performed at an illuminance of 12 mW / cm². 2 The procedure involved three light irradiations, with each irradiation lasting 10 minutes (n=2 or 3).

[0092] A significant decrease in melanin production by melanocytes (melanoma cells) was detected when the FBS concentration was greater than 0.2% but less than 5% (v / v).

[0093] (Optimal melanocyte seeding density) Figure 1 shows the evaluation results for the seeding density of melanocytes (melanoma cells) when the FBS concentration in the DMEM for melanin production induction is 2% (v / v). This evaluation was performed at an illuminance of 12 mW / cm². 2 The procedure involved three light irradiations, each lasting 10 minutes. n=2 or 3.

[0094] Seeding density is 0.1 × 10 4 cells / cm 2 Super and 2.2 x 10 4 cells / cm 2 When the value was less than [value missing], a significant decrease in melanin production by melanocytes (melanoma cells) was detected.

[0095] (Seeding density 0.8×10 4 cells / cm 2 (Evaluation results for FBS 2% (v / v)) Cell seeding density is 0.8 × 10 4 cells / cm 2 (7×10 4 Figures 2B and 2C show data on melanin production under different light conditions (see Figure 2A) when the FBS concentration in the DMEM for melanin production induction was 2% (v / v) in cells / well, 6-well plate (n=4). Figure 2B shows an illuminance of 12 mW / cm². 2 The following shows the amount of melanin produced in the groups that received light irradiation for 7.5 minutes once or 10 minutes once. Figure 2C shows the amount produced at an illuminance of 12 mW / cm². 2The following shows the amount of melanin produced in the groups that received light irradiation for 3 minutes three times, 5 minutes three times, 7.5 minutes three times, or 10 minutes three times.

[0096] As shown in Figure 2B, the illuminance is 12 mW / cm². 2 In this case, a significant decrease in melanin production (*p<0.05) was detected with light irradiation for 7.5 minutes once or once for 10 minutes compared to the control group. As shown in Figure 2C, the illuminance is 12 mW / cm². 2 A significant decrease in melanin production (*p<0.05) was detected compared to the control group after light irradiation of 5 minutes three times, 7.5 minutes three times, or 10 minutes three times.

[0097] (Suitable light irradiation conditions) Seeding density is 0.8 × 10 4 cells / cm 2 (7×10 4 cells / well (6-well plate) or 1.5 × 10 4 cells / cm 2 (14×10 4 We investigated the intracellular melanin content, protein content, and melanin production (melanin content / protein content) (%) of melanocytes (melanoma cells) under various light irradiation conditions (illuminance, irradiation time per irradiation, number of irradiations (1 or 3 times)) with the control group set to 100%, using cells / well, 6-well plates, with FBS concentration in DMEM for melanin production induction at 2% (v / v) or 4% (v / v) (n=3 or 4).

[0098] At least under the following light irradiation conditions, a significant decrease in melanin production in melanocytes compared to the control group (*p<0.05) was detected. (1) Seeding density is 0.8 × 10 4 cells / cm 2 In the case of FBS 2%(v / v) ·Illuminance: 12mW / cm 2 Irradiation time: 7.5 minutes, Number of irradiations: 1 ·Illuminance: 12mW / cm 2 Irradiation time: 10 minutes, Number of irradiations: 1 ·Illuminance: 100mW / cm2 Irradiation time: 3 minutes, Number of irradiations: 1 ·Illuminance: 12mW / cm 2 Irradiation time: 5 minutes, Number of irradiations: 1 ·Illuminance: 6.5mW / cm 2 Irradiation time: 10 minutes, Number of irradiations: 3 ·Illuminance: 12mW / cm 2、 Irradiation time: 5 minutes, Number of irradiations: 3 ·Illuminance: 12mW / cm 2 Treatment time: 7.5 minutes, Number of treatments: 3 ·Illuminance: 12mW / cm 2 Irradiation time: 10 minutes, Number of irradiations: 3 ·Illuminance: 18mW / cm 2 Irradiation time: 5 minutes, Number of irradiations: 3 times ·Illuminance: 18mW / cm 2 Irradiation time: 10 minutes, Number of irradiations: 3 ·Illuminance: 100mW / cm 2 Irradiation time: 3 minutes, Number of irradiations: 3 times ·Illuminance: 100mW / cm 2 Irradiation time: 5 minutes, Number of irradiations: 3 times (2) Seeding density is 1.5 × 10 4 cells / cm 2 In the case of FBS 4%(v / v) ·Illuminance: 12mW / cm 2 Irradiation time: 10 minutes, Number of irradiations: 3 ·Illuminance: 12mW / cm 2 Irradiation time: 20 minutes, Number of irradiations: 3 ·Illuminance: 100mW / cm 2 Irradiation time: 5 minutes, Number of irradiations: 3 times

[0099] Some of the data obtained under the above light irradiation conditions are shown in Figures 3 and 4.

[0100] As shown in the data above and Figure 3, the seeding density is 0.8 × 10 4 cells / cm 2 If the FBS concentration is 2% (v / v), the cumulative irradiation dose (mJ / cm²) 2When the (illuminance × time (seconds) × number of irradiations) was between 3,000 and 200,000 (for example, 5,400 and 180,000), a significant decrease in melanin production by melanocytes compared to the control group (*p<0.05) was detected. As shown in the data above and in Figure 4, the seeding density is 1.5 × 10 4 cells / cm 2 If the FBS concentration is 4% (v / v), the cumulative irradiation dose (mJ / cm²) 2 When the (illuminance × time (seconds) × number of irradiations) was between 3,000 and 200,000 (for example, 43,200 and 90,000), a significant decrease in melanin production by melanocytes compared to the control group (*p<0.05) was detected. Furthermore, as shown in Figures 3 and 4, the cumulative irradiation dose (mJ / cm²) 2 When the cumulative irradiation dose (mJ / cm²) was less than 180,000, a significant decrease in melanocyte protein quantity (*p<0.05) was less likely to occur, and it was easier to obtain only the effect of suppressing melanocyte production. Therefore, the cumulative irradiation dose (mJ / cm²) 2 When the ) value was between 3000 and less than 180000 (for example, 5400 to 90000), a significant decrease in melanin production by melanocytes (melanoma cells) compared to the control group (*p<0.05) was detected in a state where the melanocytes were less susceptible to the toxicity caused by light irradiation. In this embodiment, the light irradiation was performed at a wavelength of 505 nm as an example, but melanin production can be similarly evaluated with visible light, preferably green or yellow wavelengths, more preferably 490 to 570 nm (for example, 490 nm to 525 nm), or light in which any of these wavelengths is in the peak wavelength range.

[0101] Example 2. Examination of culture time Melanocyte seeding density 0.8 × 10 4 cells / cm 2 (7×10 4 Cells / well, 6-well plate), FBS concentration in DMEM for melanin production induction set to 2% (v / v), light irradiation at 505 nm (illuminance: 12 mW / cm²). 2The light irradiation was performed twice (at the timing of the first irradiation in Example 1, and 24 hours later), for 10 minutes each time. After light irradiation, the culture was extended for an additional 24 hours beyond the culture time in Example 1, and then harvested. Except for the above, the evaluation was carried out in the same manner as in Example 1 (n=2).

[0102] As shown in Figure 5, when the culture period was extended, no significant change in melanin production by melanocytes (melanoma cells) was detected in the light-irradiated group. [Industrial applicability]

[0103] The evaluation method of this embodiment can evaluate the effect of light irradiation on melanin production in melanocytes, and therefore can be suitably used to determine light irradiation conditions for suppressing or promoting melanin production in melanocytes, thus having industrial applicability.

Claims

1. Irradiating melanocytes with light, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, A method for evaluating the effects of light irradiation on melanin production in melanocytes, including [specific example].

2. The method according to claim 1, wherein the culture medium contains more than 0.2% and less than 5% (v / v) serum.

3. Light-irradiated melanocytes in the culture medium 0.1 × 10 ４ cells / cm ２ Super and 2.2 x 10 ４ cells / cm ２ The method according to claim 1 or 2, wherein the seeds are sown at a density less than 100%.

4. The method according to claim 1 or 2, wherein the light is visible light.

5. The method according to claim 2, wherein the serum is fetal bovine serum.

6. The method according to claim 2, wherein the serum concentration is 1-4% (v / v).

7. Irradiating melanocytes with light, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, When the amount of melanin produced by melanocytes decreases due to light irradiation, the conditions of that light irradiation are selected as light irradiation conditions to suppress melanin production by melanocytes. A method for determining light irradiation conditions to suppress melanin production in melanocytes, including the following.

8. Irradiating melanocytes with light, Culturing light-irradiated melanocytes in a culture medium, To quantify the amount of melanin produced by melanocytes, When the amount of melanin produced by melanocytes increases due to light irradiation, the conditions of that light irradiation are selected as light irradiation conditions to promote melanin production by melanocytes. A method for determining light irradiation conditions to promote melanin production in melanocytes, including the conditions mentioned above.

9. The method according to claim 7 or 8, wherein the culture medium contains more than 0.2% and less than 5% (v / v) serum.

10. Light-irradiated melanocytes in the culture medium 0.1 × 10 ４ cells / cm ２ Super and 2.2 x 10 ４ cells / cm ２ The method according to claim 7 or 8, wherein the seeds are sown at a density less than [amount missing].

11. The method according to claim 7 or 8, wherein the light is visible light.

12. The method according to claim 9, wherein the serum is fetal bovine serum.

13. The method according to claim 9, wherein the serum is at a concentration of 1-4% (v / v).

14. A skin treatment method (excluding medical procedures) for suppressing melanin production in the skin, comprising irradiating the skin with light under light irradiation conditions determined by the method of claim 7.

15. A skin treatment method (excluding medical procedures) for promoting melanin production in the skin, comprising irradiating the skin with light under light irradiation conditions determined by the method described in claim 8.

16. A beauty device comprising a light irradiation means for irradiating light according to light irradiation conditions determined by the method described in claim 7 or 8.

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