In-vitro melanin full-thickness skin model and construction method and application thereof

Abstract

The invention discloses an in-vitro melanin full-thickness skin model and a construction method and application thereof. The construction method comprises the following steps: improving a culture medium formula, after a corium layer is constructed, firstly adding melanocytes, then adding keratinocytes, and carrying out gas-liquid culture to obtain the in-vitro melanin full-thickness skin model. According to the construction method, when the melanocytes are in independent contact with the corium layer, the state of the melanocytes can be good, the melanocytes not only have signal interaction with the corium layer, but also have signal interaction with keratinocytes of the epidermal layer, a three-cell complete interaction system in the skin model is formed, and the three-cell interaction system is closer to real skin; and a main functional cell interaction system of each layer of real skin can be effectively simulated.

Description

Technical Field

[0001] The present application relates to the technical field of skin models, and in particular to an in vitro melanin full-thickness skin model and a construction method and application thereof. Background Art

[0002] In vitro alternative models, such as cell models and skin models, have become the mainstream experimental tools for product efficacy research in the cosmetics industry. However, in vitro cell models are two-dimensional interactive systems composed of a single cell or two or more cells. They can only reflect changes in genes, proteins, metabolism, etc. at the cellular level, and cannot simulate the three-dimensional in vitro spatial structure of the skin. In addition, the cell functions of different skin layers cannot be displayed, which limits functional research and narrows the data for cosmetic efficacy research. In vitro skin models can make up for the defects of in vitro cell models. The process of constructing skin models is that keratinocytes, melanocytes or fibroblasts in the epidermis or dermis of human skin differentiate and grow into the same tissue structure as human skin through the nutrient supply of the culture medium. It can not only retain the material and structural basis of the cell microenvironment in the body, but also show the intuitiveness and condition controllability of cell culture. It can systematically reflect the signal interaction results of each layer of cell groups after the action of cosmetics, and fully demonstrate the efficacy of cosmetics on the skin. In addition, insoluble products can be directly applied to the surface of the in vitro skin model to simulate the normal use of cosmetics by humans, so that the active substances can act on the living cell layer in the in vitro skin model through transdermal absorption to mobilize cell functions.

[0003] In traditional efficacy demonstration experiments with whitening effects, the available in vitro models include 3D melanocyte epidermal skin models and melanocyte full-thickness skin models. In the melanocyte epidermal skin model, only the melanocyte and keratinocyte systems are combined, and only the two cell population systems interact. According to literature reports, the functional effects of melanocytes are jointly regulated by keratinocytes and fibroblasts, etc. However, the melanocyte epidermal skin model can only unilaterally reflect the interaction effect of these two cells in the epidermis, lacking the functional participation of fibroblasts. The melanocyte full-thickness skin model is a skin model in which melanocytes, keratinocytes and fibroblasts are cultured and differentiated as seed cells. Melanocytes and keratinocytes are located in the basal layer, and below the basal layer is the dermis composed of fibroblasts and matrix. The behavior of melanocytes to secrete melanin is dually regulated by fibroblasts and keratinocytes.

[0004] Regarding the construction of a full-thickness melanin skin model, the traditional process is to add melanocytes and keratinocytes at the same time in a certain ratio. However, keratinocytes usually account for a large proportion, resulting in keratinocytes easily covering the dermis after inoculation of the two cells, which in turn causes the melanocytes to be unable to fully contact the dermis, resulting in weak information interaction between the melanocytes and the dermis. Summary of the invention

[0005] Based on this, it is necessary to provide an in vitro full-thickness melanin skin model and its construction method and application. The in vitro full-thickness melanin skin model includes a complete three-cell interaction system and is closer to real skin.

[0006] The first aspect of the present application provides a method for constructing an in vitro melanin full-thickness skin model, the construction method comprising the following steps:

[0007] The fibroblasts are mixed with the support carrier, and SK1 culture medium is added for submerged culture to prepare a dermis layer;

[0008] Inoculating melanocytes onto the surface of the dermis, and adding SK1 culture medium for submerged culture;

[0009] Inoculating keratinocytes onto the surface of the dermis layer loaded with the melanocytes, adding SK1 culture medium for submerged culture, forming a basal layer including the melanocytes and the keratinocytes, and obtaining a preliminary model;

[0010] The preliminary model was submerged again in SK1 medium;

[0011] The preliminary model after the submerged culture is subjected to air-liquid surface culture to prepare the in vitro melanin full-thickness skin model;

[0012] The SK1 culture medium includes DMEM / F12 mixed culture medium, basic fibroblast growth factor, recombinant human transforming growth factor, recombinant human insulin growth factor, transferrin, bovine pituitary extract, NaHCO 3 , phosphodiesterase inhibitors, and hydrocortisone.

[0013] In some embodiments, the SK1 medium further comprises one or more of fetal bovine serum, insulin, thyroxine and heparin.

[0014] In some embodiments, the SK1 medium comprises SK' medium, 15-20 μg / mL basic fibroblast growth factor, 1-5 μg / mL transferrin and 0.1-1 μg / mL hydrocortisone; wherein the SK' medium comprises DMEM / F12 mixed medium, 0.3-3 mg / mL NaHCO 3, 1-2 μg / mL recombinant human transforming growth factor, 0.05-0.10 mmol / L phosphodiesterase inhibitor, 0.15-0.2% bovine pituitary extract and 0.01-0.015 μg / mL recombinant human insulin growth factor; optionally, the SK' culture medium also includes one or more of the following components: 3-6 ng / ml insulin, 0.05-2 μg / ml thyroxine, 10% fetal bovine serum and 2-4 μg / mL heparin.

[0015] In some embodiments, in the step of gas-liquid surface culture, the culture system includes a mixed culture medium, and the mixed culture medium includes epidermal growth factor, keratinocyte growth factor and CaCl 2 and SK' medium.

[0016] In some embodiments, the mixed culture medium satisfies one or more of the following characteristics:

[0017] The concentration of the epidermal growth factor is 1 μg / mL to 15 μg / mL;

[0018] The concentration of the keratinocyte growth factor is 2 μg / mL to 8 μg / mL;

[0019] The CaCl 2 The concentration is 0.5mM~4mM.

[0020] In some embodiments, in the gas-liquid surface culturing step, the mixed culture medium includes SK2 culture medium, SK3 culture medium and SK4 culture medium.

[0021] In some embodiments, the step of performing the gas-liquid surface culture comprises: culturing the preliminary model after the submerged culture again at the gas-liquid surface of the SK2 medium; removing the SK2 medium and transferring it to the SK3 medium for gas-liquid surface culture; removing the SK3 medium and transferring it to the SK4 medium for gas-liquid surface culture; wherein the SK2 medium comprises SK' medium, 0.1-0.4 μg / mL hydrocortisone, 1-5 μg / mL epidermal growth factor, 1-2 μg / mL transferrin, 2-4 μg / mL keratinocyte growth factor, 0.5-1 mM CaCl 2 and 5-10 μg / mL basic fibroblast growth factor;

[0022] The SK3 culture medium comprises SK' culture medium, 5-10 μg / mL basic fibroblast growth factor, 5-10 μg / mL epidermal growth factor, 2-4 μg / mL transferrin, 4-6 μg / mL keratinocyte growth factor, 1-2 mM CaCl 2and 0.1-0.4 μg / mL hydrocortisone;

[0023] The SK4 culture medium comprises SK' culture medium, 5-10 μg / mL basic fibroblast growth factor, 0.1-0.4 μg / mL hydrocortisone, 10-15 μg / mL epidermal growth factor, 4-8 μg / mL transferrin, 2-8 μg / mL keratinocyte growth factor, 2-4 mM CaCl 2 .

[0024] In some embodiments, during any submerged culture, the culture medium is replaced every day.

[0025] In some embodiments, in the step of mixing the fibroblasts with the support carrier and performing submerged culture, the submerged culture time is 7 to 8 days.

[0026] In some embodiments, in the step of inoculating melanocytes onto the surface of the dermis and culturing them underwater, the underwater culturing time is 1 to 2 days.

[0027] In some embodiments, in the step of inoculating keratinocytes onto the surface of the dermis layer loaded with the melanocytes and performing submerged culture, the submerged culture time is 2 to 3 days.

[0028] In some embodiments, in the step of submerging the preliminary model in SK1 medium again, the time of submerged culture is 1 to 2 days.

[0029] In some embodiments, in the step of performing the gas-liquid interface culture, the time of gas-liquid interface culture in the SK2 culture medium is 2 to 4 days, the time of gas-liquid interface culture in the SK3 culture medium is 2 to 3 days, and the time of gas-liquid interface culture in the SK4 culture medium is 3 to 5 days.

[0030] In some embodiments, the support carrier is a polymer scaffold having interconnected micropores inside, and at least a portion of the fibroblasts are located on the microporous surface of the polymer scaffold.

[0031] In some specific embodiments, the polymer scaffold is composed of a biocompatible material, which is a synthetic polymer including one or more of polystyrene and polycaprolactone.

[0032] In some embodiments, the polymer stent is selected from at least one of the following stents: ALvetex stent, Biotek 3D Insert TM PS scaffold.

[0033] In some embodiments, in the step of mixing fibroblasts with the polymer scaffold, a fibroblast suspension is added to the interior of the polymer scaffold, and the density of fibroblasts in the fibroblast suspension is 1×10 6 / mL~1.5×10 6 The seeding density of the fibroblasts relative to the surface of the polymer scaffold is 4.2×10 5 Pieces / cm 2 ~6.0×10 5 Pieces / cm 2 .

[0034] In some embodiments, in the step of inoculating melanocytes onto the surface of the dermis, a melanocyte suspension is added to the surface of the dermis, and the density of melanocytes in the melanocyte suspension is 1.5×10 6 / mL~2×10 6 The seeding density of the melanocytes relative to the surface of the dermis is 3.8×10 5 Pieces / cm 2 ~5.5×10 5 Pieces / cm 2 .

[0035] In some embodiments, in the step of seeding keratinocytes onto the surface of the dermis layer loaded with the melanocytes, a keratinocyte suspension is added to the surface of the dermis layer loaded with the melanocytes, and the keratinocyte density in the keratinocyte suspension is 1.5×10 6 / mL~2×10 6 The seeding density of the keratinocytes relative to the surface of the dermis loaded with the melanocytes is 4×10 5 Pieces / cm 2 ~6.0×10 5 Pieces / cm 2 .

[0036] In some embodiments, the seeding ratio of the keratinocytes, the melanocytes, and the fibroblasts is (1.3-1.6):(1.3-1.6):1.

[0037] The second aspect of the present application provides an in vitro melanin full-thickness skin model, which is constructed using the construction method of the in vitro melanin full-thickness skin model described in the first aspect of the present application.

[0038] In some embodiments, the in vitro melanin full-thickness skin model includes a dermis layer and a basal layer stacked in sequence; wherein the dermis layer includes a polymer scaffold and fibroblasts, the interior of the polymer scaffold has interconnected micropores, and at least a portion of the fibroblasts are located on the microporous surface of the polymer scaffold; the basal layer includes melanocytes and keratinocytes, and the melanocytes are located between the fibroblasts and the keratinocytes.

[0039] The third aspect of the present application provides a UVR-induced in vitro full-thickness melanin skin model, wherein the in vitro full-thickness melanin skin model described in the second aspect of the present application is subjected to UVR induction, wherein the UVR induction refers to ultraviolet radiation induction.

[0040] In some embodiments, the UVR induction includes UVA induction, wherein the UVA induction refers to the induction of 2 ~40mJ / cm 2 induced under the conditions of .

[0041] In some embodiments, the UVR induction includes UVB induction, wherein the UVB induction refers to the induction of 2 ~60mJ / cm 2 induced under the conditions of .

[0042] In some embodiments, the UVR-inducing conditions include an energy of 30 mJ / cm 2 ~60mJ / cm 2 The induction time is 9 to 10 days.

[0043] The fourth aspect of the present application provides the use of the in vitro melanin full-thickness skin model prepared by the construction method of the in vitro melanin full-thickness skin described in the first aspect, the in vitro melanin full-thickness skin model described in the second aspect, or the UVR-induced in vitro melanin full-thickness skin model described in the third aspect in the efficacy testing of cosmetics.

[0044] In some embodiments, the efficacy of the cosmetic includes at least one of whitening and firming and anti-wrinkle.

[0045] In some embodiments, the cosmetics include cosmetics having whitening efficacy or firming and anti-wrinkle efficacy.

[0046] In the construction method of the in vitro melanin full-thickness skin model provided in the present application, the culture medium formula is improved so that the melanocytes can be in good condition when they are in contact with the dermis alone. After the dermis is constructed, the melanocytes are added first and then the keratinocytes. The melanocytes can completely contact the keratinocytes and fibroblasts at the same time, which significantly increases the interaction between the melanocytes and the fibroblasts in the dermis and positions the melanocytes in the basal layer. The positioning of the melanocytes in the skin model is closer to the real skin. A large number of melanocytes not only have signal interactions with the dermis, but also interact with the signals of the keratinocytes in the epidermis, forming a complete three-cell interaction system in the skin model, which is closer to the real skin and can effectively simulate the main functional cell interaction systems of each layer of the real skin.

[0047] Furthermore, the polymer scaffold can be used as the sole support carrier of dermal cells, and the dermis can be constructed using the polymer scaffold and fibroblasts without introducing animal-derived collagen or other types of collagen. This allows the formation of the dermal extracellular matrix (ECM) to be completed by the extracellular assembly of ECM-related proteins secreted by fibroblasts, which is closer to the formation mechanism of the ECM in the dermis of human skin.

[0048] In addition, the culture medium formula in the culture system for air-liquid interface culture has been improved (using SK2 culture medium, SK3 culture medium and SK4 culture medium), which is conducive to constructing a system that more realistically simulates the interaction between the main functional cells of each layer of real skin.

[0049] In the in vitro melanin full-thickness skin model constructed using the aforementioned in vitro melanin full-thickness skin model construction method, a large number of melanocytes not only have signal interactions with the dermis, but also interact with the signals of the keratinocytes in the epidermis, forming a three-cell complete interaction system in the skin model, which is closer to real skin. The in vitro melanin full-thickness skin model is more applicable to whitening efficacy research. Furthermore, the dermis layer uses a polymer scaffold as a supporting carrier for fibroblasts, and further, animal-derived collagen and other types of collagen can be omitted, so that the formation of the dermis extracellular matrix (ECM) is formed by extracellular assembly of ECM-related proteins secreted by fibroblasts, which is closer to the formation mechanism of the dermis ECM of real skin. When the in vitro melanin full-thickness skin model is constructed using seed cells derived from human skin, the in vitro melanin full-thickness skin model can be used as an in vitro melanin human full-thickness skin model.

[0050] The in vitro melanin full-thickness skin model provided above can be applied to cosmetic efficacy testing, and can provide a good experimental application model for exploring the interaction between melanocytes, keratinocytes and fibroblasts at the in vitro skin model level for cosmetics with whitening or firming and anti-wrinkle effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0052] Figure 1 A is an apparent image of a full-thickness melanin skin model after UVR irradiation in one embodiment of the present application; B is a corresponding silver staining result image of a section of a full-thickness melanin skin model after UVR irradiation;

[0053] Figure 2 A is an apparent image of a full-thickness melanin skin model without UVR irradiation in one embodiment of the present application; B is a silver staining result image of a corresponding section of a full-thickness melanin skin model without UVR treatment;

[0054] Figure 3 This is a silver staining result diagram of a section of a full-thickness skin model of in vitro melanin stimulated by UVR in one embodiment of the present application, wherein the box is the papillary area, and melanin can be observed in the dermis;

[0055] Figure 4 This is the silver staining result of human skin in vitro after UVR stimulation. The box is the papillary area, and melanin can be observed in the dermis.

[0056] Figure 5 A is an apparent image of a full-thickness melanin skin model stimulated by UVR that was not cultured with the modified SK1 medium in Comparative Example 1; B is a silver staining result image of a section of a full-thickness melanin skin model stimulated by UVR that was not cultured with the modified SK1 medium;

[0057] Figure 6 This is a silver staining result of a section of a full-thickness melanin skin model constructed by simultaneous inoculation of melanocytes and keratinocytes in Comparative Example 2. DETAILED DESCRIPTION

[0058] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0060] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0061] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0062] The terms "having", "containing", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0063] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0064] In this application, if the unit of the data range is followed only by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 1-2 μg / mL means that the units of the left endpoint "1" and the right endpoint "2" are both μg / mL, which have the same meaning as 1 μg / mL-2 μg / mL. In addition, similar descriptions involving other parameters also apply to the above understanding.

[0065] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0066] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0067] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0068] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0069] In this application, unless otherwise specified, "in vitro melanin full-thickness skin model" refers to a three-dimensional or 3D in vitro model that simulates a full-thickness skin structure containing melanocytes, wherein "full-thickness" refers to at least the basal layer and the dermis, and the "in vitro melanin full-thickness skin model" is a skin model that uses cells including melanocytes, keratinocytes and fibroblasts as seed cells for culture and / or differentiation. Among them, the construction of the "dermis" includes the culture and / or differentiation of fibroblasts, and the construction of the "basal layer" includes the culture and / or differentiation of melanocytes and keratinocytes.

[0070] In this application, unless otherwise specified, the term "melanocyte" used also refers to melanocytes, which are specialized cells present in the basal layer of the epidermis of the skin and are mainly responsible for the production of melanin.

[0071] In this application, unless otherwise specified, the term "keratinocyte" used also refers to keratinocytes, which are the main cells of the epidermis and can constitute the epidermis together with melanocytes. Keratinocytes can produce keratin. During the differentiation process, keratinocytes can continuously move upward and produce tough keratin, eventually forming a complete skin protective layer - the stratum corneum.

[0072] In this application, unless otherwise specified, the term "culture system" used refers to a system used to culture cells in a skin model, which may be a basal culture medium that meets the basic growth requirements of the cells; or it may be a liquid system containing at least a basal culture medium, for example, in addition to the basal culture medium, nutrients such as growth factors may be added according to the growth requirements of the cultured cells.

[0073] There have been some reports on the construction of a full-thickness skin model of melanin, using animal-derived collagen as a model dermis construction material mixed with fibroblasts to construct the dermis, but the operation process is complicated. There are also some reports that after the dermis is constructed, the traditional process is to add melanocytes and keratinocytes at the same time in a certain ratio, and usually keratinocytes account for a large proportion. After inoculating the two cells, keratinocytes easily cover the dermis, which easily leads to melanocytes being unable to fully contact the dermis, resulting in the blocking of the information interaction performance between melanocytes and the dermis being weak. Based on this, the present application at least provides an in vitro full-thickness skin model of melanin and its construction method and application, and the in vitro full-thickness skin model of melanin includes a complete three-cell interaction system, which is closer to real skin.

[0074] In some embodiments of the present application, in the process of constructing a full-thickness melanin skin model in vitro, a polymer scaffold is used as a carrier of the dermis layer, and fibroblasts are added to construct the dermis layer, thereby avoiding the use of animal collagen and various prefabricated collagens, improving operation efficiency, and simplifying experimental operations. Furthermore, after the dermis layer is constructed, melanocytes are added first, and then keratinocytes are added, so that the melanocytes have sufficient contact with the dermis layer, and a complete signal interaction is established between the melanocytes and the fibroblasts of the dermis layer, thereby promoting the interaction between keratinocytes and fibroblasts and melanocytes, forming a complete three-cell interaction system, which can more realistically simulate the main functional cell interaction system of each layer of real human skin.

[0075] The construction of the in vitro melanin full-thickness skin model provided in this application can not only meet the application needs of exploring whitening effects such as dermal pigmentation tests, but also meet the application needs of anti-wrinkle effects such as exploring the effects of cell interactions on skin aging.

[0076] The first aspect of the present application provides a method for constructing an in vitro melanin full-thickness skin model.

[0077] In some embodiments, the method for constructing an in vitro melanin full-thickness skin model comprises the following steps:

[0078] S100: Mix the fibroblasts with the support carrier, add SK1 culture medium for submerged culture, and prepare the dermis layer;

[0079] S200: Melanocytes were inoculated onto the surface of the dermis and SK1 medium was added for submerged culture;

[0080] S300: Keratinocytes were inoculated onto the surface of the dermis loaded with melanocytes, and SK1 culture medium was added for submerged culture to form a basal layer including melanocytes and keratinocytes, thereby obtaining a preliminary model;

[0081] S400: The preliminary model was cultured again in SK1 medium;

[0082] S500: The preliminary model after the submerged culture is cultured again by air-liquid surface culture to obtain an in vitro melanin full-thickness skin model;

[0083] SK1 medium includes DMEM / F12 mixed medium, basic fibroblast growth factor, recombinant human transforming growth factor, recombinant human insulin growth factor, transferrin, bovine pituitary extract, NaHCO 3 , phosphodiesterase inhibitors and hydrocortisone, wherein the transferrin can be bovine transferrin.

[0084] In some specific embodiments, the SK1 medium may further comprise one or more of fetal bovine serum (FBS), insulin, thyroxine and heparin.

[0085] In the construction method of the in vitro full-thickness melanin skin model provided above, the culture medium formula is improved so that the melanocytes can be in good condition when they are in contact with the dermis alone. After the dermis is constructed, melanocytes are added first and then keratinocytes, which significantly increases the interaction between melanocytes and dermal fibroblasts and positions the melanocytes in the basal layer. The positioning of melanocytes in the skin model is closer to real skin. A large number of melanocytes not only interact with the dermis, but also interact with the signals of keratinocytes in the epidermis, forming a complete three-cell interaction system in the skin model, which is closer to real skin and can effectively simulate the main functional cell interaction systems of each layer of real skin.

[0086] In the present application, unless otherwise specified, the "dermis layer" in the in vitro melanin full-thickness skin model at least includes a supporting carrier and fibroblasts, which are used to simulate the dermis layer in real skin.

[0087] In the present application, unless otherwise specified, the "basal layer" in the in vitro melanin full-thickness skin model includes at least melanocytes and keratinocytes, and is used to simulate the epidermis in real skin.

[0088] In the present invention, unless otherwise specified, "submerged culture" refers to a method of culturing cells by inoculating them in a liquid culture medium.

[0089] In the present invention, unless otherwise specified, "air-liquid surface culture" refers to a culture method in which one side of the cells is in contact with a liquid culture medium and the other side is exposed to air.

[0090] In some embodiments, the support carrier is a polymer scaffold.

[0091] In some embodiments, the method for constructing an in vitro melanin full-thickness skin model comprises the following steps:

[0092] S100': Using the polymer scaffold as a support carrier, fibroblasts were seeded into the internal micropores of the polymer scaffold, and SK1 culture medium was added for submerged culture to obtain the dermis;

[0093] S200': Melanocytes were inoculated onto the surface of the dermis and SK1 medium was added for submerged culture;

[0094] S300': Keratinocytes were inoculated onto the surface of the dermis loaded with melanocytes, and SK1 culture medium was added for submerged culture to form a basal layer including melanocytes and keratinocytes, thus obtaining a preliminary model;

[0095] S400': The preliminary model was cultured again in SK1 medium;

[0096] S500': the preliminary model after the submerged culture is cultured again by air-liquid surface culture to obtain an in vitro melanin full-thickness skin model;

[0097] SK1 medium includes DMEM / F12 mixed medium, basic fibroblast growth factor, recombinant human transforming growth factor, recombinant human insulin growth factor, transferrin, bovine pituitary extract, NaHCO 3 , phosphodiesterase inhibitors and hydrocortisone, wherein the transferrin can be bovine transferrin.

[0098] In some specific embodiments, the SK1 medium may further comprise one or more of fetal bovine serum (FBS), insulin, thyroxine and heparin.

[0099] Polymer scaffolds can be used as the sole support carrier for dermal cells, and the dermis can be constructed using polymer scaffolds and fibroblasts without introducing animal-derived collagen or other types of collagen. This allows the formation of the dermal extracellular matrix (ECM) to be completed by the extracellular assembly of ECM-related proteins secreted by fibroblasts, which is closer to the formation mechanism of the ECM in the dermis of human skin.

[0100] In some embodiments, fibroblasts, melanocytes and keratinocytes are separated and cultured before constructing an in vitro melanin full-thickness skin model. The separation and culture can be performed using existing technical means in the art, including but not limited to conventional technical means.

[0101] In some embodiments, fibroblasts are obtained by isolating and culturing primary fibroblasts; melanocytes are obtained by isolating and culturing primary melanocytes; and keratinocytes are obtained by isolating and culturing primary keratinocytes. In addition, the polymer scaffold can be pre-treated before constructing an in vitro melanin full-thickness skin model to improve the compatibility between the polymer scaffold and the fibroblasts. In a non-limiting manner, the polymer scaffold can be pre-treated with a 70% to 75% (v / v) ethanol aqueous solution.

[0102] In the present application, "DMEM / F12 mixed medium" is the conventional DMEM / F12 medium in the art, which is a combination of DMEM medium and F12 medium, used for cell culture, and the volume ratio of DMEM to F12 is 1:1. The DMEM / F12 mixed medium in the present application was purchased from Gibco.

[0103] In some embodiments, the SK1 medium includes SK' medium, 15-20 μg / mL basic fibroblast growth factor, 1-5 μg / mL transferrin and 0.1-1 μg / mL hydrocortisone; wherein the SK' medium includes DMEM / F12 mixed medium, 0.3-3 mg / mL NaHCO 3 , 1-2 μg / mL recombinant human transforming growth factor, 0.05-0.10 mmol / L phosphodiesterase inhibitor, 0.15-0.2% bovine pituitary extract and 0.01-0.015 μg / mL recombinant human insulin growth factor; further optionally, the SK' medium also includes one or more of the following components: 3-6 ng / ml insulin, 0.05-2 μg / ml thyroxine, 10% fetal bovine serum and 2-4 μg / mL heparin.

[0104] It is understood that, in some embodiments, the concentration of basic fibroblast growth factor in SK1 culture medium can be but is not limited to 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL or 20 μg / mL, or a value or range between any two of the above values.

[0105] In some embodiments, the concentration of transferrin in the SK1 medium may be, but is not limited to, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL or 5 μg / mL, or a value or range between any two of the above values.

[0106] In some embodiments, the concentration of hydrocortisone in SK1 medium can be, but is not limited to, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL or 1 μg / mL, or a value or range between any two of the above values.

[0107] In some embodiments, the concentration of recombinant human transforming growth factor in SK' medium can be but is not limited to 1 μg / mL, 1.2 μg / mL, 1.4 μg / mL, 1.6 μg / mL, 1.8 μg / mL or 2 μg / mL, or a value or range between any two of the above values.

[0108] In some embodiments, the concentration of recombinant human insulin growth factor in SK' medium can be but is not limited to 0.01 μg / mL, 0.011 μg / mL, 0.012 μg / mL, 0.013 μg / mL, 0.014 μg / mL or 0.015 μg / mL, or a value or range between any two of the above values.

[0109] In some embodiments, the mass fraction of the bovine pituitary extract in the SK' medium may be, but is not limited to, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2% or a value or range between any two of the above values.

[0110] In some embodiments, the concentration of the phosphodiesterase inhibitor in SK' medium can be, but is not limited to, 0.05mmoL / L, 0.06mmoL / L, 0.07mmoL / L, 0.08mmoL / L, 0.09mmoL / L or 0.1mmoL / L, or a value or range between any two of the above values.

[0111] In some embodiments, the concentration of insulin in the SK' medium can be, but is not limited to, 3 ng / mL, 4 ng / mL, 5 ng / mL, or 6 ng / mL, or a value or range between any two of the above values.

[0112] In some embodiments, the concentration of thyroxine in SK' medium can be, but is not limited to, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, 1.8 μg / mL, 1.9 μg / mL or 2 μg / mL, or a value or range between any two of the above values.

[0113] In some embodiments, the concentration of heparin in SK' medium can be, but is not limited to, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL or 4 μg / mL, or a value or range between any two of the above values.

[0114] It should be noted that in some embodiments, in the step of gas-liquid surface culture, the culture system includes a mixed culture medium, and the mixed culture medium includes epidermal growth factor, keratinocyte growth factor and CaCl 2 At least one of the above and SK' medium. Among them, the concentration of epidermal growth factor can be 1μg / mL to 15μg / mL; the concentration of keratinocyte growth factor can be 2μg / mL to 8μg / mL; CaCl 2 The concentration can be 0.5mM to 4mM. It is understandable that re-submerging the preliminary model with SK1 culture medium helps maintain the function and activity of fibroblasts in the dermis, allowing them to secrete and self-assemble ECM-related proteins and promote the formation of ECM; it also helps maintain the vitality of primary melanocytes and keratinocytes, so that the cells are in a stable state of vitality after entering the new culture system, ensuring the normal performance of their cell biological functions. Subsequently, through gas-liquid surface culture of the mixed culture medium, the prepared in vitro melanin full-thickness skin model can be accurately positioned in each layer, and the interaction performance between cells in each layer is excellent.

[0115] In some embodiments, the mixed culture medium may include SK2 culture medium, SK3 culture medium and SK4 culture medium. In other words, the culture system in the above steps 500 and 500' may include SK1 culture medium, SK2 culture medium, SK3 culture medium and SK4 culture medium; the step of performing air-liquid surface culture includes: culturing the preliminary model after the second underwater culture in the air-liquid surface of SK2 culture medium, removing the SK2 culture medium, and transferring it to the air-liquid surface of SK3 culture medium; removing the SK3 culture medium, and transferring it to the air-liquid surface of SK4 culture medium to prepare an in vitro melanin full-thickness skin model.

[0116] Among them, SK2 medium includes SK' medium, 0.1-0.4 μg / mL hydrocortisone, 1-5 μg / mL epidermal growth factor, 1-2 μg / mL transferrin, 2-4 μg / mL keratinocyte growth factor, 0.5-1 mM CaCl 2 and 5-10 μg / mL basic fibroblast growth factor; SK3 medium includes SK' medium, 5-10 μg / mL basic fibroblast growth factor, 5-10 μg / mL epidermal growth factor, 2-4 μg / mL transferrin, 4-6 μg / mL keratinocyte growth factor, 1-2 mM CaCl 2 and 0.1-0.4 μg / mL hydrocortisone; SK4 medium includes SK' medium, 5-10 μg / mL basic fibroblast growth factor, 0.1-0.4 μg / mL hydrocortisone, 10-15 μg / mL epidermal growth factor, 4-8 μg / mL transferrin, 2-8 μg / mL keratinocyte growth factor, and 2-4 mM CaCl 2 .

[0117] It should be noted that, in some embodiments, the time for gas-liquid surface culture in SK2 medium is 2 to 4 days, the time for gas-liquid surface culture in SK3 medium is 2 to 3 days, and the time for gas-liquid surface culture in SK4 medium is 3 to 5 days.

[0118] In some embodiments, the concentration of hydrocortisone in SK2 medium, SK3 medium, and SK4 medium may be, but is not limited to, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, or a value or range between any two of the above values.

[0119] In some embodiments, the concentration of basic fibroblast growth factor in SK2 medium, SK3 medium, and SK4 medium can be 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, or a value or range between any two of the above values.

[0120] In some embodiments, the concentration of epidermal growth factor in SK2 medium can be, but is not limited to, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, or a value or range between any two of the above values.

[0121] In some embodiments, the concentration of transferrin in the SK2 medium can be, but is not limited to, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, or a value or range between any two of the above values.

[0122] In some embodiments, the concentration of keratinocyte growth factor in SK2 medium can be, but is not limited to, 2 μg / mL, 3 μg / mL, 4 μg / mL, or a value or range between any two of the above values.

[0123] In some embodiments, CaCl in SK2 medium 2 The concentration of may be, but is not limited to, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM or a value or range between any two of the above values.

[0124] In some embodiments, the concentration of epidermal growth factor in SK3 medium can be, but is not limited to, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, or a value or range between any two of the above values.

[0125] In some embodiments, the concentration of transferrin in the SK3 medium can be, but is not limited to, 2 μg / mL, 3 μg / mL, 4 μg / mL, or a value or range between any two of the above values.

[0126] In some embodiments, the concentration of keratinocyte growth factor in SK3 medium can be, but is not limited to, 4 μg / mL, 5 μg / mL, 6 μg / mL, or a value or range between any two of the above values.

[0127] In some embodiments, CaCl in SK3 medium 2 The concentration can be, but is not limited to, 1 mM, 1.5 mM, 2 mM or a value or range between any two of the above values.

[0128] In some embodiments, the concentration of epidermal growth factor in SK4 medium can be but is not limited to 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL or 15 μg / mL, or a value or range between any two of the above values.

[0129] In some embodiments, the concentration of transferrin in the SK4 medium can be, but is not limited to, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, or a value or range between any two of the above values.

[0130] In some embodiments, the concentration of keratinocyte growth factor in SK4 medium may be, but is not limited to, 6 μg / mL, 7 μg / mL or 8 μg / mL, or a value or range between any two of the above values.

[0131] In some embodiments, CaCl in SK4 medium 2 The concentration can be, but is not limited to, 2 mM, 2.5 mM, 3 mM, 3.5 mM or 4 mM, or a value or range between any two of the above values.

[0132] In some embodiments, in the step of culturing the fibroblasts and the polymer scaffold in a mixed manner, the culturing time is 7 days, 8 days, or any time period in between the two time periods.

[0133] In some embodiments, in the step of inoculating melanocytes onto the surface of the dermis for culturing, the culturing time is 1 day, 2 days or any time period in between the above two time periods.

[0134] In some embodiments, in the step of inoculating keratinocytes onto the surface of the dermis layer loaded with melanocytes and culturing them, the culturing time is 2 days, 3 days or any time in between the above two time periods.

[0135] In some embodiments, in the step of submerging the preliminary model in SK1 medium again, the submerged culture period is 1 day, 2 days, or any period in between.

[0136] In some embodiments, in the step of constructing an in vitro melanin full-thickness skin model, it is necessary to replace the culture medium every day.

[0137] In the process of constructing the aforementioned in vitro melanin full-thickness skin model, the culture medium formula for air-liquid interface culture was improved (using SK2 culture medium, SK3 culture medium and SK4 culture medium), which is conducive to constructing a more realistic simulation of the main functional cell interaction system of each layer of real skin.

[0138] In some embodiments, in the step of mixing the fibroblasts with the polymer scaffold, a fibroblast suspension is added to the interior of the polymer scaffold, and the density of the fibroblasts in the fibroblast suspension is 1×10 6 / mL~1.5×10 6 Without limitation, the density of fibroblasts in the fibroblast suspension may be, but is not limited to, 1×10 6 / mL, 1.05×10 6 / mL, 1.1×10 6 / mL, 1.15×10 6 / mL, 1.2×10 6 / mL, 1.25×10 6 / mL, 1.3×10 6 / mL, 1.35×10 6 / mL, 1.4×10 6 / mL, 1.45×10 6 / mL or 1.5×10 6 The seeding density of the fibroblasts relative to the surface of the polymer scaffold is 4.2×10 5 Pieces / cm 2 ~6.0×10 5 Pieces / cm 2 Without limitation, the seeding density of fibroblasts relative to the surface of the polymer scaffold may be, but is not limited to, 4.2×10 5 Pieces / cm 2 , 4.4×10 5 Pieces / cm 2 , 4.6×10 5 Pieces / cm 2 , 4.8×10 5 Pieces / cm 2 , 5×10 5 Pieces / cm 2 , 5.2×10 5 Pieces / cm 2 , 5.4×10 5 Pieces / cm 2 , 5.6×10 5 Pieces / cm 2 , 5.8×10 5 Pieces / cm 2 , 6×10 5 Pieces / cm 2 Or a value or range between any two of the above values.

[0139] In some embodiments, in the step of inoculating melanocytes onto the surface of the dermis, a melanocyte suspension is added to the surface of the dermis, and the density of melanocytes in the melanocyte suspension is 1.5×10 6 / mL~2×10 6 / mL; the seeding density of melanocytes relative to the surface of the dermis is 3.8×10 5 Pieces / cm 2 ~5.5×10 5 / cm2 Without limitation, the density of melanocytes in the melanocyte suspension may be, but is not limited to, 1.5×10 6 / mL, 1.6×10 6 / mL, 1.7×10 6 / mL, 1.8×10 6 / mL, 1.9×10 6 / mL, 2.0×10 6 / mL or a value or range between any two of the above values; the seeding density of melanocytes relative to the surface of the dermis can be but is not limited to 3.8×10 5 Pieces / cm 2 , 4×10 5 Pieces / cm 2 4.2×10 5 Pieces / cm 2 , 4.4×10 5 Pieces / cm 2 , 4.6×10 5 Pieces / cm 2 , 4.8×10 5 Pieces / cm 2 , 5×10 5 Pieces / cm 2 , 5.2×10 5 Pieces / cm 2 , 5.5×10 5 Pieces / cm 2 Or a value or range between any two of the above values.

[0140] In some embodiments, in the step of seeding keratinocytes onto the surface of the dermis layer loaded with melanocytes, a keratinocyte suspension is added to the surface of the dermis layer loaded with melanocytes, and the keratinocyte density in the keratinocyte suspension is 1.5×10 6 / mL~2×10 6 The seeding density of keratinocytes on the surface of the dermis loaded with melanocytes was 4×10 5 Pieces / cm 2 ~6.0×10 5 Pieces / cm 2 Without limitation, the density of keratinocytes in the keratinocyte suspension may be, but is not limited to, 1.5×10 6 / mL, 1.6×10 6 / mL, 1.7×10 6 / mL, 1.8×10 6 / mL, 1.9×10 6 / mL, 2.0×10 6 / mL or a value or range between any two of the above values; the seeding density of keratinocytes relative to the surface of the dermis loaded with melanocytes can be, but is not limited to, 4×10 5 Pieces / cm 2 4.2×10 5 Pieces / cm 2 , 4.4×10 5 Pieces / cm 2 , 4.6×10 5 Pieces / cm 2 , 4.8×10 5 Pieces / cm 2 , 5×10 5 Pieces / cm 2 , 5.2×10 5 Pieces / cm 2 , 5.4×10 5 Pieces / cm 2 , 5.6×10 5 Pieces / cm 2 , 5.8×10 5 Pieces / cm 2 , 6×10 5 Pieces / cm 2 Or a value or range between any two of the above values.

[0141] In some embodiments, the inoculation ratio of keratinocytes, melanocytes, and fibroblasts is (1.3-1.6):(1.3-1.6):1; further, the inoculation ratio of keratinocytes, melanocytes, and fibroblasts is 1.4:1.4:1.

[0142] Whether the above-mentioned in vitro melanin full-thickness skin model is successfully constructed can be verified or evaluated by the following method: subjecting the constructed in vitro melanin full-thickness skin model to UVR stimulation, and then staining and observing or photographing it. If the melanin content of the model is observed to increase, it indicates that the in vitro melanin full-thickness skin model is successfully constructed. The staining method can be, but is not limited to, Masson-Fontana staining, HE staining, and section silver staining.

[0143] The second aspect of the present application provides an in vitro melanin full-thickness skin model, which is constructed using the construction method of the first aspect of the present application.

[0144] In some embodiments, an in vitro melanin full-thickness skin model is provided, which includes a dermis layer and a basal layer stacked in sequence; wherein the dermis layer includes a supporting carrier and fibroblasts, the interior of the supporting carrier has interconnected micropores, and at least a portion of the fibroblasts are located on the microporous surface of the supporting carrier; the basal layer includes melanocytes and keratinocytes, and the melanocytes are located between the fibroblasts and the keratinocytes.

[0145] In this application, unless otherwise specified, the "support carrier" used to construct the dermis layer refers to a biocompatible scaffold having interconnected micropores inside, and having a suitable porosity and a suitable internal surface area to allow dermal cells (such as fibroblasts) to attach, grow and interact.

[0146] In some embodiments, the support carrier is a polymer scaffold.

[0147] In some embodiments, the in vitro melanin full-thickness skin model includes a dermis layer and a basal layer stacked in sequence: wherein the dermis layer includes a polymer scaffold and fibroblasts, the interior of the polymer scaffold has interconnected micropores, and at least a portion of the fibroblasts are located in the pores of the polymer scaffold; the basal layer includes melanocytes and keratinocytes, and the melanocytes are located between the fibroblasts and the keratinocytes.

[0148] In the in vitro melanocyte full-thickness skin model constructed using the aforementioned construction method, a large number of melanocytes not only interact with the dermis, but also interact with the keratinocytes in the epidermis, forming a complete three-cell interaction system in the skin model, which is closer to real skin. This in vitro melanocyte full-thickness skin model is more applicable to whitening efficacy research.

[0149] In the present application, unless otherwise specified, the "in vitro melanin full-thickness skin model includes a dermis layer and a basal layer stacked in sequence" means that the layers of the skin model are stacked and interlocked with each other, for example, the dermis layer and the basal layer are tightly stacked together, and each layer has its own unique function and composition.

[0150] In this application, unless otherwise specified, "polymer scaffold" refers to a scaffold made of a biocompatible polymer material, which has interconnected micropores inside, high porosity and a large internal surface area, which is conducive to the attachment, growth and interaction of cells. Generally, the average diameter of the pores of the polymer scaffold is 42 microns.

[0151] In this application, unless otherwise specified, "at least a portion" means more than 90% of the number of added cells, for example, more than 90% of the fibroblast content is located in the pores of the polymer scaffold. In this application, unless otherwise specified, when "above" is used to describe a value, it includes the value itself. For example, more than 90% means greater than or equal to 90%.

[0152] In some embodiments, the polymer scaffold is composed of a biocompatible material, and the biocompatible material includes a synthetic polymer. Without limitation, the synthetic polymer may include one or more of polystyrene and polycaprolactone.

[0153] The dermis in the in vitro melanin full-thickness skin model can use a polymer scaffold as a carrier of fibroblasts, and further, animal-derived collagen and other types of collagen can be omitted, so that the formation of the extracellular matrix (ECM) of the dermis is all formed by the extracellular assembly of ECM-related proteins secreted by fibroblasts, which is closer to the formation mechanism of the ECM of the dermis of real skin. When the in vitro melanin full-thickness skin model is constructed using seed cells derived from human skin, the in vitro melanin full-thickness skin model can be used as an in vitro melanin human full-thickness skin model.

[0154] In some embodiments, the polymer stent is selected from at least one of the following: ALvetex stent, Biotek 3D Insert TM PS scaffold.

[0155] In this application, unless otherwise specified, "ALvetex scaffold" refers to a technical product used for three-dimensional cell culture, usually made of polystyrene (PS) or similar biocompatible materials, with high porosity and large surface area, which is conducive to cell attachment, growth and interaction.

[0156] The definition of UVR may refer to the third aspect of this application.

[0157] The third aspect of the present application provides a UVR-induced in vitro full-thickness melanin skin model.

[0158] The UVR-induced in vitro full-thickness melanin skin model can be obtained by subjecting the in vitro full-thickness melanin skin model of the second aspect of the present application to UVR induction.

[0159] In the present application, unless otherwise specified, "UVR induction" refers to ultraviolet radiation (UVR). Ultraviolet rays can be irradiated on the in vitro melanin full-thickness skin model of the second aspect of the present application to obtain a UVR-induced in vitro melanin full-thickness skin model. UVR can include two types: UVA (320-400nm) and UVB (290-320nm), wherein UVA uses ultraviolet rays with a wavelength of 320-400nm for radiation, and UVB uses ultraviolet rays with a wavelength of 290-320nm for radiation.

[0160] In some embodiments, the energy of UVA in UVR induction may be, but is not limited to, 30 mJ / cm 2 、31mJ / cm 2 、32mJ / cm 2 、33mJ / cm 2 、34mJ / cm 2 、35mJ / cm 2 、36mJ / cm 2 、37mJ / cm 2、38mJ / cm 2 、39mJ / cm 2 or 40mJ / cm 2 , or a value or range between any two of the above values.

[0161] In some embodiments, the energy of UVB in UVR induction can be, but is not limited to, 50 mJ / cm 2 、51mJ / cm 2 、52mJ / cm 2 、53mJ / cm 2 、54mJ / cm 2 、55mJ / cm 2 、56mJ / cm 2 、57mJ / cm 2 、58mJ / cm 2 、59mJ / cm 2 or 60mJ / cm 2 , or a value or range between any two of the above values.

[0162] In some embodiments, UVR induction may be UVA irradiation only, UVB irradiation only, or may include UVA and UVB irradiation.

[0163] In some embodiments, the UVR induction period is 9 days, 10 days, or any period therebetween.

[0164] The fourth aspect of the present application provides the application of the in vitro full-thickness melanin skin model constructed by the construction method of the first aspect of the present application, the in vitro full-thickness melanin skin model of the second aspect of the present application, or the UVR-induced in vitro full-thickness melanin skin model of the third aspect of the present application in the efficacy testing of cosmetics.

[0165] In some embodiments, the efficacy of the cosmetic includes at least one of whitening and firming and anti-wrinkle.

[0166] In some embodiments, the cosmetics include cosmetics having whitening efficacy or firming and anti-wrinkle efficacy.

[0167] The in vitro melanin full-thickness skin model provided above can be applied to cosmetic efficacy testing, and can provide a good experimental application model for exploring the interaction between melanocytes, keratinocytes and fibroblasts at the in vitro skin model level for cosmetics with whitening or anti-wrinkle effects.

[0168] Some examples are provided below.

[0169] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0170] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0171] In the following examples, unless otherwise specified, "double antibody" refers to a mixture of penicillin and streptomycin, which has a well-known meaning in the art. Taking "1% double antibody" as an example, it refers to double antibody accounting for 1% of the total mass of the solution, wherein the concentration of penicillin is 100U / mL and the concentration of streptomycin is 0.1mg / mL. The double antibody in the examples of this application was purchased from Purnosai, with the item number PB180120.

[0172] In the following examples, unless otherwise specified, "dispase" refers to a non-specific metalloproteinase that can separate single cells from various tissues or organs for the separation of primary cells and cell passage, and has a well-known meaning in the art, including dispase II (Dispase II) and the like. The dispase in the examples of the present application is dispase II (Dispase II), which was purchased from Shanghai Maokang Biotechnology Co., Ltd. Among them, the concentration of dispase is 0.6-2.4U / mL, and U is defined as: the amount of enzyme required to convert a certain amount of substrate into a product within a certain period of time under certain conditions.

[0173] Example 1: Construction of an in vitro melanin full-thickness skin model

[0174] This application provides a human melanin full-thickness skin model construction system, which is achieved through the following technical solutions:

[0175] (1) Isolation and culture of primary keratinocytes: Place human skin tissue in a culture dish and wash it seven times with a 4°C precooled PBS solution containing double antibodies (1% double antibodies, double antibodies refer to a combination of penicillin and streptomycin). Stop washing when there is no blood on the skin surface and in the washing solution. Carefully cut off the subcutaneous tissue with ophthalmic scissors and wash it four times with a 4°C precooled PBS solution containing double antibodies (1% double antibodies, double antibodies refer to penicillin and streptomycin). Use scissors to cut the tissue block into 1×2 mm 2The size of the cells was added to 2U / mL of dispase digestion solution and digested overnight (24h) at 4°C. The dermis and epidermis were separated using surgical forceps, and the separated epidermal tissue was placed in a culture dish. After digestion for 30min at 37°C with 0.25% trypsin (containing 0.2mg / mL of ethylenediaminetetraacetic acid (EDTA)), the digestion was terminated with DMEM culture medium containing 10% fetal bovine serum, and the cells were filtered with a 200-mesh sieve, centrifuged, and the supernatant was collected to collect the precipitated cells. The cell pellet was washed and resuspended with PBS, and the cells were resuspended with serum-free culture medium for keratinocytes and inoculated into a T75 flask at 37°C and 5% CO 2 Culture in an incubator, change the medium every 24 hours, and subculture when the cells grow to a plating rate of about 80%-85%. The cells can be expanded to the third to fifth generations for model construction.

[0176] The serum-free keratinocyte culture medium: human epidermal keratinocyte culture medium KC-Growth, added with 0.012 μg / mL recombinant human insulin growth factor, 0.16 μg / mL hydrocortisone, 0.21 ng / mL human epidermal growth factor, 6 μg / mL bovine transferrin, and 0.18% by mass of bovine pituitary extract (purchased from Aibixin, abs9119).

[0177] (2) Isolation and culture of primary melanocytes: Place human skin tissue in a culture dish and wash it seven times with a PBS solution containing double antibody (1% double antibody) precooled at 4°C. Stop washing when there is no blood on the skin surface and in the washing solution. Carefully cut off the subcutaneous tissue with ophthalmic scissors and wash it four times with a PBS solution containing double antibody (1% double antibody) precooled at 4°C. Use scissors to cut the tissue into 1×2 mm 2 The size of the cells was added to 2U / mL of dispase digestion solution and digested overnight (24h) at 4°C. The dermis and epidermis were separated using surgical forceps, and the separated epidermal tissue was placed in a culture dish. After digestion with 0.25% trypsin (containing 0.2mg / mL EDTA) digestion solution at 37°C for 30min, the digestion was terminated with DMEM culture medium containing 10% fetal bovine serum, and the supernatant was collected after filtering with a 200-mesh sieve. The cell pellet was washed and resuspended with PBS, and the cells were resuspended with melanocyte culture medium and inoculated into a T75 flask at 37°C and 5% CO 2 Culture in an incubator, change the medium every 24 hours, and subculture when the cells grow to a plating rate of about 80%-85%. The cells can be expanded to the third to fifth generations for model construction.

[0178] The above-mentioned melanocyte culture medium: M254 culture medium (purchased from Life Technologies, USA), 0.5% fetal bovine serum by mass fraction, 5ng / mL basic fibroblast growth factor, 0.4% bovine pituitary extract by mass fraction, 3μg / mL heparin, 0.24μg / mL hydrocortisone, 0.08μg / mL recombinant human insulin-like growth factor-I, 3μg / mL transferrin and 5nM endothelin-1.

[0179] (3) Isolation and culture of primary fibroblasts: Place human skin tissue in a culture dish and wash it seven times with a 4°C precooled PBS solution containing double antibody (1% double antibody). Stop washing when there is no blood on the skin surface and in the washing solution. Carefully cut off the subcutaneous tissue with ophthalmic scissors and wash it four times with a 4°C precooled PBS solution containing double antibody (1% double antibody). Use scissors to cut the tissue into 1×2 mm 2 The size of the cells was added to 2U / mL of dispase digestion solution and digested overnight (24h) at 4°C. The dermis and epidermis were separated using surgical forceps, and the separated dermal tissue was placed in a culture dish. After digestion for 30min at 37°C with 0.25% trypsin (containing 0.2mg / mL EDTA), the digestion was terminated with DMEM culture medium containing 10% fetal bovine serum, and the cells were filtered with a 200-mesh sieve, centrifuged, and the supernatant was collected to collect the precipitated cells. The cell pellet was washed and resuspended with PBS, and the cells were resuspended with fibroblast culture medium and inoculated into a T75 flask at 37°C and 5% CO. 2 Culture in an incubator, change the medium every 24 hours, and subculture when the cells grow to a plating rate of about 80%-85%. The cells can be expanded to the third to fifth generations for model construction.

[0180] The above-mentioned fibroblast culture medium: 5 ng / mL insulin, 1 μg / mL thyroxine, 18 μg / mL basic fibroblast growth factor and 1.5 μg / mL recombinant human transforming growth factor (TGF) were added to the DMEM / F12 mixed culture medium.

[0181] (4) Scaffold treatment: Prepare 70% anhydrous ethanol and filter it using a 0.22 μm filter membrane. Treat each scaffold with 5.5 mL of anhydrous ethanol. Take a new 6-well plate, add 5 mL of 70% ethanol to each well, transfer the scaffold to this 6-well plate under a sterile environment, and immerse it for 10 minutes; after transferring it to a new 6-well plate, wash it with PBS three times, 5 mL each time, and wash it for 5 minutes each time. Discard PBS, add 5.5 mL of SK1 culture medium to each well, and place it at 37°C with 5% CO 2 The incubator is ready for use.

[0182] The above-mentioned scaffold is ALvetex scaffold (purchased from Reprocell, AVP004-12), which is a technical product for three-dimensional cell culture. It is made of polystyrene (PS) or similar biocompatible materials, with high porosity and large surface area, which is conducive to cell attachment, growth and interaction. ALvetex scaffold is a polymer material composed of microporous structure, which allows cells to grow inside the scaffold to form a three-dimensional structure, helping to form an extracellular matrix (ECM)-like environment, thereby mimicking the complexity and structure of in vivo tissues. In addition, the above-mentioned scaffold can also be used 3D Biotek3D Insert TM PS scaffold.

[0183] The above SK1 medium: DMEM / F12 mixed medium with 10% fetal bovine serum (FBS), 5 ng / mL insulin, 1 μg / mL thyroxine, 18 μg / mL basic fibroblast growth factor, 1.5 μg / mL recombinant human transforming growth factor (TGF), 1.5 mg / mL NaHCO 3 , 0.08mmol / L phosphodiesterase inhibitor (IBMX), 0.012μg / mL recombinant human insulin growth factor, 3μg / mL bovine transferrin, 0.18% bovine pituitary extract, 3μg / mL heparin and 0.5μg / mL hydrocortisone.

[0184] (5) Construction of dermis: The fibroblasts obtained in step (3) were cultured in SK1 medium and the cell density was adjusted to 1.25 × 10 6 / mL, take 0.8mL of cell suspension into the scaffold treated in step (4), and the seeding density of fibroblasts on the surface of the polymer scaffold is 5×10 5 Pieces / cm 2 , 5.5 mL of SK1 culture medium was added to the 6-well plate and cultured for 7 days, and the culture medium was replaced every day.

[0185] (6) Melanocyte addition: The melanocytes obtained in step (2) were added to adjust the cell density to 1.75×10 6 Take 0.8 mL of cell suspension onto the dermis constructed in (5), slowly add it, and evenly spread it. The seeding density of melanocytes relative to the surface of the dermis is 5×10 5 Pieces / cm 2 . Add 6 mL of SK1 culture medium into the 6-well plate and culture for 1 day.

[0186] (7) Addition of keratinocytes: The keratinocytes obtained in step (1) were added to adjust the cell density to 1.75×10 6 / mL, take 0.8mL of cell suspension to the upper part of the model (6) that already contains melanocytes and dermis, slowly add, and evenly spread. The seeding density of keratinocytes relative to the surface of the dermis loaded with the melanocytes is 5.5×10 5 Pieces / cm 2 5.5 mL of SK1 culture medium was added to the 6-well plate and cultured for 2 days, with the culture medium replaced every day to obtain a preliminary model.

[0187] (8) Submerged culture again: The preliminary model was placed in a 6-well plate and 5.5 mL of SK1 culture medium was added for submerged culture for 2 days.

[0188] (9) Air-liquid surface culture: aspirate the SK1 medium in the 6-well plate in step (8), add 4.5 mL of SK2 medium to each well, and culture at the air-liquid surface for 2 days; aspirate the SK2 medium, add 4.5 mL of SK3 medium to each well, and culture at the air-liquid surface for 3 days; aspirate the SK4 medium, add 4.5 mL of SK3 medium to each well, and culture at the air-liquid surface for 4 days to obtain an in vitro full-thickness melanin skin model.

[0189] The above SK2 medium: DMEM / F12 mixed medium with 10% FBS, 5 ng / mL insulin, 1 μg / mL thyroxine, 8 μg / mL basic fibroblast growth factor, 1.5 μg / mL recombinant human transforming growth factor (TGF), 1.5 mg / mL NaHCO 3 , 0.08mmol / L phosphodiesterase inhibitor (IBMX), 0.012μg / mL recombinant human insulin growth factor, 0.18% bovine pituitary extract, 2μg / mL heparin, 0.2μg / mL hydrocortisone, 1μg / mL epidermal growth factor, 1μg / mL transferrin, 2μg / mL keratinocyte growth factor and 0.5mM CaCl 2 .

[0190] SK3 medium: DMEM / F12 mixed medium with 10% FBS, 5 ng / mL insulin, 1 μg / mL thyroxine, 8 μg / mL basic fibroblast growth factor, 1.5 μg / mL recombinant human transforming growth factor (TGF), and 1.5 mg / mL NaHCO 3 , 0.08mmol / L phosphodiesterase inhibitor (IBMX), 0.012μg / mL recombinant human insulin growth factor, 0.18% bovine pituitary extract, 2μg / mL heparin, 0.2μg / mL hydrocortisone, 5μg / mL epidermal growth factor, 2μg / mL transferrin, 4μg / mL keratinocyte growth factor and 1mM CaCl 2 .

[0191] SK4 medium: DMEM / F12 mixed medium with 10% FBS, 5 ng / mL insulin, 1 μg / mL thyroxine, 8 μg / mL basic fibroblast growth factor, 1.5 μg / mL recombinant human transforming growth factor (TGF), and 1.5 mg / mL NaHCO 3 , 0.08mmol / L phosphodiesterase inhibitor (IBMX), 0.012μg / mL recombinant human insulin growth factor, 0.18% bovine pituitary extract, 2μg / mL heparin, 0.2μg / mL hydrocortisone, 10μg / mL epidermal growth factor, 4μg / mL transferrin, 6μg / mL keratinocyte growth factor and 2mM CaCl 2 .

[0192] (10) Whitening efficacy application model establishment process:

[0193] Construction of UVR-induced melanin full-thickness skin model: The in vitro melanin full-thickness skin model was placed in a 6-well plate supplemented with 5.5 mL of SK1 culture medium and the in vitro melanin full-thickness skin model was irradiated with UVA 30 mJ / cm 2 and UVB 50mJ / cm 2 Irradiation was continued for 9 days, and SK1 medium was replaced every day. After completion, the model was collected for subsequent experiments.

[0194] Performance Validation Testing of an In Vitro Melanin Full-Thickness Skin Model

[0195] The UVR-induced in vitro melanin full-thickness skin model (UVR-induced group) and the in vitro melanin full-thickness skin model without UVR irradiation (non-UVR-induced group) constructed in Example 1 were photographed by a Nikon (D7500) camera. The photographic results can be seen in Figure 1 A and Figure 2 A in . It should be noted that Figure 1 The surface of the skin model of the UVR-induced group shown in A shows wrinkles, which is caused by the stress response under UVR stimulation, and is similar to the clinical symptoms of normal human skin wrinkling and then peeling caused by high-dose sunlight exposure.

[0196] Furthermore, the UVR-induced in vitro melanin full-thickness skin model (UVR-induced group) and the melanin full-thickness skin model without UVR irradiation (non-UVR-induced group) constructed in Example 1 were sectioned and silver-stained, respectively. Figure 1 B and Figure 2 B. The steps of silver staining the slices of the in vitro melanin full-thickness skin model are as follows:

[0197] The Masson-Fontana melanin kit (purchased from Shaanxi Yike Biotechnology Service Co., Ltd.) was used to stain melanin and observe the UVR-stimulated in vitro melanin full-thickness skin model.

[0198] 1. Fixation: Select an appropriate fixative, such as 10% neutral formalin or 4% paraformaldehyde, to fix the in vitro melanin full-thickness skin model tissue (1 h). The fixative used in this application is 10% neutral formalin.

[0199] 2. Sectioning: Dehydrate the fixed tissue (ethanol gradient dehydration), wax-immerse and embed (paraffin embedding), and then cut into paraffin sections of appropriate thickness.

[0200] 3. Dewaxing of paraffin sections: put the sections into xylene I (purchased from Sinopharm Chemical Reagent Co., Ltd.) for the first dewaxing for 8 minutes, xylene II for the second dewaxing for 8 minutes, anhydrous ethanol I for the first dehydration for 6 minutes, anhydrous ethanol II for the second dehydration for 6 minutes, 95% alcohol for 6 minutes, 85% alcohol for 6 minutes, 75% alcohol for 5 minutes, and rinse with running water;

[0201] 4. Add Fontana ammonia silver solution and immerse for 1 hour at 37°C. After the time is up, observe the melanin staining under the microscope;

[0202] 5. Wash with distilled water twice, 3 minutes each time;

[0203] 6. Add the Hypo solution dropwise at room temperature for 5 minutes;

[0204] 7. Wash with distilled water twice, 5 min each time;

[0205] 8. Add neutral red solution at room temperature and counterstain for 15 minutes;

[0206] 9. Wash with water after dyeing until the excess dye is gone;

[0207] 10. Add 95% alcohol for 10 seconds and anhydrous ethanol II for 5 seconds, and wash until the slide is transparent;

[0208] 11. Add xylene III for 5 min, xylene II for 5 min, and xylene I for 5 min;

[0209] 12. Seal the slides with neutral gum (purchased from Shaanxi Yike Biotechnology Service Co., Ltd.).

[0210] The difference between the above Ⅰ, Ⅱ and Ⅲ lies mainly in the use stage and time of the solution. There is no difference in concentration. Since they cannot be used again after use, Ⅰ, Ⅱ and Ⅲ are used to distinguish them.

[0211] Compared with the non-UVR induced group ( Figure 2 A and Figure 2 Compared with the UVR-induced group ( Figure 1 A and Figure 1 B) The skin model in Example 1 is darker and the melanin content in the skin model is higher, indicating that the full-thickness melanin skin model constructed in Example 1 has a good response to UVR stimulation. The full-thickness melanin skin model becomes darker due to the secretion of a large amount of melanin by melanocytes under UVR stimulation, which is the same as the mechanism of human skin becoming darker after being stimulated by UVR. It also further indicates that the in vitro full-thickness melanin skin model prepared in Example 1 can simulate the scene of human skin becoming darker after being stimulated by UVR, and further obtain the UVR-induced in vitro full-thickness melanin skin model.

[0212] In order to further evaluate the performance of the full-thickness skin model prepared in Example 1 in terms of application, the UVR-stimulated human ex vivo skin was silver-stained (the silver staining results of human ex vivo skin are shown in FIG. Figure 4 ), and compared with the silver-stained sections of the UVR-stimulated in vitro melanin full-thickness skin model prepared in Example 1. Figure 3 This is the silver staining result of the slice of the full-thickness skin model stimulated by UVR in vitro. Figure 3 and Figure 1 B in FIG. 1 is taken from a different area of ​​the same slice.

[0213] like Figure 4 As shown in the figure, after UVR irradiation, melanin appears in both the epidermis and dermis of human skin ex vivo. This is because the keratinocytes in the epidermis and the fibroblasts in the dermis secrete related factors (such as MMP-1, etc.), which causes the dermal-epidermal junction (DEJ) to be destroyed. Melanocytes secrete a large amount of melanin, and part of the melanin enters the keratinocytes. As the epidermis differentiates into the stratum corneum, the other part of the melanin is destroyed. Large pores appear between the epidermis and the melanin secreted by the melanocytes pass through the pores to the dermis. The fibroblasts in the dermis will engulf the melanin, forming melanin accumulation in the dermis. In addition, from Figure 4 It can be seen that there are both papillary areas and flat DEJ areas in real human skin tissue, which is consistent with Figure 1 Figure B and Figure 3 The results are similar to those of , indicating that the in vitro melanin full-thickness skin model constructed in this application has formed a complete three-cell interaction system, the dermis and epidermis have a signal exchange system, and when stimulated by UVR, an interaction system of keratinocytes, melanocytes, and fibroblasts is formed.

[0214] Comparative Example 1: In vitro melanin skin model prepared using unmodified SK1 culture medium

[0215] The in vitro melanin skin model after UVR induced stimulation was prepared by a method substantially the same as that in Example 1, except that: in steps (4) to (9), the SK1 medium was replaced with an unmodified SK1 medium to culture the melanin skin model. The medium formula of the unmodified SK1 medium was as follows: 10% FBS, 5 ng / mL insulin, 1 μg / mL thyroxine, 8 μg / mL basic fibroblast growth factor, 0.8 μg / mL recombinant human transforming growth factor (TGF), 1.5 mg / mL NaHCO were added to the DMEM / F12 mixed medium. 3 , 0.008 μg / mL recombinant human insulin growth factor, 1.5 μg / mL bovine transferrin and 0.18% bovine pituitary extract.

[0216] The in vitro melanin skin model of Comparative Example 1 was induced by UVR to obtain the UVR-induced in vitro melanin skin model (unmodified SK1 culture medium group) for surface photography. The photographic results can be seen in Figure 5 A. Further, the UVR-induced in vitro melanin skin model (unmodified SK1 culture medium group) of Comparative Example 1 was silver stained, see Figure 5 B in.

[0217] Compared with the unmodified SK1 medium group ( Figure 5 A and Figure 5 Compared with the modified SK1 medium group ( Figure 1 A and Figure 1 B) The skin model is darker and contains more melanin, indicating that the modified SK1 culture medium is more effective in maintaining the function of melanocytes in the in vitro full-thickness melanoma skin model, and melanocytes secrete melanin normally, so that the in vitro full-thickness melanoma skin model has complete functional effects. It should be noted that when the unmodified SK1 culture medium is used to culture the melanoma skin model in steps (4)-(8), and only the unmodified SK1 culture medium is used for gas-liquid surface culture in step (9), the amount of melanin in the UVR-induced in vitro melanoma skin model obtained is less than the accumulated amount of melanin in the UVR-induced in vitro melanoma skin model obtained in Comparative Example 1, indicating that in the process of preparing the in vitro full-thickness melanoma skin model, the selection of the culture medium is very important, which affects the signal transmission of each layer of cells in the in vitro full-thickness melanoma skin model, and thus affects the accuracy of the construction of the in vitro full-thickness melanoma skin model.

[0218] Comparative Example 2: An in vitro melanin skin model prepared by simultaneously inoculating melanocytes and keratinocytes.

[0219] The method is basically the same as that in Example 1, except that: in the steps of Example 1, after the dermis layer is constructed, the melanocytes and keratinocytes are mixed and inoculated on the surface of the dermis layer for cultivation to construct an in vitro melanin skin model. The ratio of melanocytes to keratinocytes is consistent with the ratio of melanocytes to keratinocytes inoculated in Example 1.

[0220] The in vitro melanin skin model prepared in Comparative Example 2 was sectioned and silver stained. The staining results were as follows: Figure 6 As shown, Figure 2 By comparing with B in the figure, it can be seen that in the in vitro melanin skin model of comparative example 2, the melanin content is low and the epidermis of the model has abnormal growth, indicating that the in vitro melanin skin model obtained by mixing melanocytes and keratinocytes and inoculating them on the surface of the dermis for cultivation has poor cell-to-cell interaction performance and cannot simulate the actual skin more realistically.

[0221] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0222] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. A method for constructing an in vitro melanin full-thickness skin model, characterized in that: The construction method comprises the following steps: The fibroblasts are mixed with the support carrier, and SK1 culture medium is added for submerged culture to prepare a dermis layer; Inoculating melanocytes onto the surface of the dermis, and adding SK1 culture medium for submerged culture; Inoculating keratinocytes onto the surface of the dermis layer loaded with the melanocytes, adding SK1 culture medium for submerged culture, forming a basal layer including the melanocytes and the keratinocytes, and obtaining a preliminary model; The preliminary model was submerged again in SK1 medium; The preliminary model after the submerged culture is subjected to air-liquid surface culture to prepare the in vitro melanin full-thickness skin model; The SK1 culture medium includes DMEM / F12 mixed culture medium, basic fibroblast growth factor, recombinant human transforming growth factor, recombinant human insulin growth factor, transferrin, bovine pituitary extract, NaHCO3, phosphodiesterase inhibitor and hydrocortisone; optionally, the SK1 culture medium also contains one or more of fetal bovine serum, insulin, thyroxine and heparin.

2. The method for constructing an in vitro melanin full-thickness skin model according to claim 1, characterized in that: The SK1 culture medium includes SK' culture medium, 15-20 μg / mL basic fibroblast growth factor, 1-5 μg / mL transferrin and 0.1-1 μg / mL hydrocortisone; wherein the SK' culture medium includes DMEM / F12 mixed culture medium, 0.3-3 mg / mL NaHCO3, 1-2 μg / mL recombinant human transforming growth factor, 0.05-0.10 mmol / L phosphodiesterase inhibitor, 0.15-0.2% bovine pituitary extract by mass fraction and 0.01-0.015 μg / mL recombinant human insulin growth factor; Optionally, the SK' medium further comprises one or more of the following components: 3-6 ng / mL insulin, 0.05-2 μg / mL thyroxine, 10% fetal bovine serum by mass, and 2-4 μg / mL heparin.

3. The method for constructing an in vitro melanin full-thickness skin model according to claim 2, characterized in that: In the step of gas-liquid surface culture, the culture system includes a mixed culture medium, and the mixed culture medium includes at least one of epidermal growth factor, keratinocyte growth factor and CaCl2 and the SK' culture medium; optionally, the mixed culture medium satisfies one or more of the following characteristics: The concentration of the epidermal growth factor is 1 μg / mL to 15 μg / mL; The concentration of the keratinocyte growth factor is 2 μg / mL to 8 μg / mL; The concentration of CaCl2 is 0.5mM to 4mM.

4. The method for constructing an in vitro melanin full-thickness skin model according to claim 3, characterized in that: In the gas-liquid surface culture step, the mixed culture medium includes SK2 culture medium, SK3 culture medium and SK4 culture medium; The steps of performing the gas-liquid surface culture include: culturing the preliminary model after the submerged culture again in the gas-liquid surface of the SK2 culture medium; removing the SK2 culture medium and transferring it to the gas-liquid surface of the SK3 culture medium; removing the SK3 culture medium and transferring it to the gas-liquid surface of the SK4 culture medium; wherein, The SK2 culture medium comprises SK' culture medium, 0.1-0.4 μg / mL hydrocortisone, 1-5 μg / mL epidermal growth factor, 1-2 μg / mL transferrin, 2-4 μg / mL keratinocyte growth factor, 0.5-1 mM CaCl2 and 5-10 μg / mL basic fibroblast growth factor; The SK3 culture medium comprises SK' culture medium, 5-10 μg / ml basic fibroblast growth factor, 5-10 μg / mL epidermal growth factor, 2-4 μg / mL transferrin, 4-6 μg / mL keratinocyte growth factor, 1-2 mM CaCl2 and 0.1-0.4 μg / mL hydrocortisone; The SK4 culture medium comprises SK' culture medium, 5-10 μg / mL basic fibroblast growth factor, 0.1-0.4 μg / mL hydrocortisone, 10-15 μg / mL epidermal growth factor, 4-8 μg / mL transferrin, 2-8 μg / mL keratinocyte growth factor and 2-4 mM CaCl2.

5. The method for constructing an in vitro melanin full-thickness skin model according to claim 4, characterized in that: Meet one or more of the following characteristics: During any submerged culture, the culture medium was changed every day; In the step of mixing the fibroblasts with the support carrier and adding SK1 culture medium for submerged culture, the submerged culture time is 7 to 8 days; In the step of inoculating melanocytes onto the surface of the dermis and adding SK1 culture medium for submerged culture, the submerged culture time is 1 to 2 days; In the step of inoculating keratinocytes onto the surface of the dermis layer loaded with the melanocytes, and adding SK1 culture medium for submerged culture, the submerged culture time is 2 to 3 days; In the step of submerging the preliminary model in SK1 medium again, the submerged culture time is 1 to 2 days; In the step of performing the gas-liquid interface culture, the time of gas-liquid interface culture in the SK2 culture medium is 2 to 4 days, the time of gas-liquid interface culture in the SK3 culture medium is 2 to 3 days, and the time of gas-liquid interface culture in the SK4 culture medium is 3 to 5 days.

6. The method for constructing an in vitro melanin full-thickness skin model according to claim 1, characterized in that: The support carrier is a polymer scaffold, the interior of the polymer scaffold has interconnected micropores, and at least a portion of the fibroblasts are located on the micropore surface of the polymer scaffold; Optionally, the polymer stent is composed of a biocompatible material, the biocompatible material is a synthetic polymer, and the synthetic polymer includes one or more of polystyrene and polycaprolactone; Further optionally, the polymer stent is selected from at least one of the following stents: ALvetex stent, Biotek 3D Insert TM PS scaffold.

7. The method for constructing an in vitro melanin full-thickness skin model according to any one of claims 1 to 6, characterized in that: Satisfies one or more of the following characteristics (a)-(d): (a) In the step of mixing fibroblasts with the polymer scaffold, a fibroblast suspension is added to the interior of the polymer scaffold, and the density of fibroblasts in the fibroblast suspension is 1×10 6 / mL~1.5×10 6 The seeding density of the fibroblasts relative to the surface of the polymer scaffold is 4.2×10 5 Pieces / cm 2 ~6.0×10 5 Pieces / cm 2 ; (b) in the step of inoculating melanocytes onto the surface of the dermis, a melanocyte suspension is added to the surface of the dermis, wherein the density of melanocytes in the melanocyte suspension is 1.5×10 6 / mL~2×10 6 The seeding density of the melanocytes relative to the surface of the dermis is 3.8×10 5 Pieces / cm 2 ~5.5×10 5 Pieces / cm 2 ; (c) in the step of inoculating keratinocytes onto the surface of the dermis layer loaded with the melanocytes, a keratinocyte suspension is added to the surface of the dermis layer loaded with the melanocytes, wherein the keratinocyte density in the keratinocyte suspension is 1.5×10 6 / mL~2×10 6 The seeding density of the keratinocytes relative to the surface of the dermis loaded with the melanocytes is 4×10 5 Pieces / cm 2 ~6.0×10 5 Pieces / cm 2 ; (d) The inoculation ratio of the keratinocytes, the melanocytes and the fibroblasts is (1.3-1.6):(1.3-1.6):

1.

8. An in vitro melanin full-thickness skin model, characterized in that: The in vitro melanin full-thickness skin model is constructed by the construction method of any one of claims 1 to 7; the in vitro melanin full-thickness skin model comprises a dermis layer and a basal layer stacked in sequence; wherein the dermis layer comprises a polymer scaffold and fibroblasts, the interior of the polymer scaffold has interconnected micropores, and at least a portion of the fibroblasts are located on the microporous surface of the polymer scaffold; the basal layer comprises melanocytes and keratinocytes, and the melanocytes are located between the fibroblasts and the keratinocytes.

9. UVR-induced in vitro full-thickness melanin skin model, characterized in that: The in vitro melanin full-thickness skin model of claim 8 is subjected to UVR induction, wherein the UVR induction refers to ultraviolet radiation induction; Optionally, the UVR induction satisfies one or more of the following characteristics: The UVR induction includes UVA induction, wherein the UVA induction refers to an induction with an energy of 30 mJ / cm 2 ~40mJ / cm 2 Induction under the conditions of The UVR induction includes UVB induction, wherein the UVB induction refers to an induction with an energy of 50 mJ / cm 2 ~60mJ / cm 2 Induction under the conditions of The UVR induction conditions include an energy of 30 mJ / cm 2 ~60mJ / cm 2 The induction time is 9 to 10 days.

10. Use of the in vitro melanin full-thickness skin model constructed by the construction method according to any one of claims 1 to 7, the in vitro melanin full-thickness skin model according to claim 8, or the UVR-induced in vitro melanin full-thickness skin model according to claim 9 in the efficacy testing of cosmetics; Optionally, the cosmetic has at least one of whitening and firming and anti-wrinkle effects; Optionally, the cosmetics include cosmetics with whitening effects or firming and anti-wrinkle effects.

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