RECONSTRUCTED SKIN MODEL

Abstract

The present invention relates to a reconstituted skin model comprising 1) a skin substitute and 2) a microbial component and a lipid component on the surface of said skin substitute, as well as a method for obtaining the reconstituted skin model. The reconstituted skin model can, in particular, be used in a screening method for the in vitro identification of an active ingredient or cosmetic formulation to prevent and / or improve at least one skin disorder induced by at least one exogenous stress; a method for evaluating the in vitro efficacy of an active ingredient or cosmetic formulation to prevent and / or treat at least one skin disorder induced by at least one exogenous stress; or an in vitro method for evaluating the tolerability of an active ingredient or cosmetic formulation.

Description

Title of the invention: RECONSTITUTED SKIN MODEL Scope of the invention

[0001] The field of the invention relates to a novel skin model incorporating a microbial component and a lipid component. This model is particularly suitable for studying and developing new active ingredients / products in the dermo-cosmetic field. Indeed, physical or chemical stress can be added to this integrated model to reproduce lesions or pathological conditions that can cause imbalances in the skin microbiota. State of the art

[0002] The skin is much more than an external covering; it is, in fact, a true organ essential to life. It constitutes a site of exchange between our internal and external environments and is therefore exposed to numerous aggressions and variations to which it constantly adapts. The skin thus performs, among other things, a true barrier function, formerly attributed solely to its structure and composition. The skin is composed of cells grouped together to form a tissue that is both resistant and flexible. These cells are distributed in three layers: the hypodermis, the dermis, and the epidermis. The hypodermis is the deepest layer of the skin. It is made up of white adipose tissue that protects the body from shocks, constitutes an energy reserve, and regulates body temperature. The dermis is the layer between the hypodermis and the epidermis. It is the thickest layer of the skin.The dermis contains blood and lymphatic vessels, nerves, sweat glands that produce sweat, and pilosebaceous follicles. It is primarily composed of macromolecules, fibroblasts, and immune system cells. The epidermis is the thinnest layer of skin. It is composed of 90% keratinocytes, which synthesize keratin and, through their differentiation, provide the skin with impermeability and protection.The epidermis is composed of four main layers: the basal layer (stratum basale), made up of keratinocytes with large nuclei, which ensure the constant renewal of the epidermis; the spinous layer (stratum spinosum), composed of large keratinocytes that gradually flatten towards the upper layers; the granular layer (stratum granulosum), composed of flattened keratinocytes that transform keratinocytes into corneocytes (anucleate cells); and finally, the stratum corneum, the outermost layer composed of corneocytes that will shed the epidermis. The epidermis is not only composed of corneocytes but also of lipids between these different cells. They are organized into lipid bilayers. The main classes of these lipids are ceramides, cholesterol, and free fatty acids. They partially ensure the skin's barrier function.

[0003] Sebum is a hydrolipidic film produced by the sebaceous glands. It is secreted onto the skin's surface where it forms a protective layer and also serves as a substrate for the skin microbiota. Sebum is predominantly lipophilic and anaerobic, and therefore helps to preserve lipophilic and anaerobic microbial species. It also protects the skin by moisturizing it and acidifying it to regulate the skin's pH.

[0004] The skin microbiota also plays a key role in the skin's protective functions. It also participates in regulating the skin's pH. The skin microbiota, also called skin flora, is located in the epidermis. It consists of microorganisms such as bacteria, viruses, fungi, and parasites. The skin microbiota evolves with age, reaching an average of 1 trillion bacteria and 1,000 species of viruses, parasites, and fungi. For bacteria, the best-known and most studied strains are Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. Some are aerobic and therefore live with the supply of oxygen, while others are anaerobic and live deeper in the stratum corneum without oxygen. Some of these bacteria produce fatty acids, others antimicrobial peptides, and even in some cases amino acids.Most of these bacteria use lipids from sebum to grow and produce these compounds.

[0005] The skin microbiota varies both quantitatively and qualitatively from one person to another. Variables such as age, sex, immune system, pH, temperature, and humidity can modify the composition of the skin microbiota. For example, the use of cosmetic or pharmaceutical products creates differences. However, two distinct categories can be identified within the skin microbiota: the "resident" flora and the "transient" flora.

[0006] As its name suggests, the resident skin flora lives in total symbiosis with the skin. These are called commensal organisms. It is important for defense against pathogenic microorganisms, because the space it occupies saturates the area and prevents the attachment of undesirable organisms.

[0007] Transient flora does not establish itself permanently on the skin surface; it varies throughout the day, depending on activities performed and changes in environmental conditions. It may be present for a few hours or a few days. The microorganisms that compose it are mostly harmless, known as saprophytes. This flora can also consist of opportunistic pathogenic bacteria and cause disease if the host's defenses are weakened. One of the most common transient species is S. aureus, implicated in atopic dermatitis. (DA).

[0008] The microorganisms present on the skin and comprising the resident flora are not opportunistic; they perform very useful functions for the protection of the body. Indeed, the skin microbiota plays various roles in maintaining the integrity of our skin. It constitutes a barrier by regulating skin homeostasis, providing it with nutrients, and competing with pathogens. For example, S. epidermidis secretes antimicrobial peptides against S. aureus. Corynebacterium striatum and C. acnes, for their part, inhibit the expression of virulence genes of the latter (Sanford and Gallo, Seminars in Immunology, 25, pp370-7, 2013). The symbiosis between host and microorganisms is essential for healthy skin. When this balance is disrupted, it is called dysbiosis.Numerous studies have shown that dysbiosis is correlated with the development of certain inflammatory skin conditions, such as acne, atopic dermatitis, and eczema. Understanding the interactions between the skin microbiota and the skin is therefore crucial in the development of new cosmetic and pharmaceutical treatments.

[0009] Today, the evaluation of cosmetic and pharmacological agents can be carried out, in particular, on in vitro reconstructed human skin models. These models contain a differentiated epidermal barrier and reflect the morphological conditions of in vivo human skin. Among the reconstructed human skin models, Van Der Krieken and his team developed a stratum comeum model from keratinocytes taken from the heels of volunteers (Acta Derm Venereol. 2016; 96(7): 873-879). A microbiota is added to the keratinocytes in suspension. However, the composition of these skin models still differs considerably from the actual composition of human skin. In particular, it does not include lipids that play a protective role for the skin and the microbiota and that are secreted by the sebaceous glands. Furthermore, it does not contain viable keratinocytes.Thus, the interaction between microorganisms and keratinocytes (secretion of cytokines, antimicrobial peptides, etc.) is not observable.

[0010] 3D models of reconstructed epidermis containing a microbial compound have also been used to study the interactions between the microbiota and the skin (Rademacher et al., Experimental Dermatology, 27, pp. 489–494, 2018). However, most of these models include colonization by a single species of microorganism, such as C. amycolatum, and therefore do not reflect the actual conditions of our skin. The presence of lipids in healthy skin models is also not described.

[0011] Consequently, there is always a need for a model that can reproduce conditions as close as possible to natural skin, preferably obtainable from easy and at a reduced cost.

[0012] Advantageously, the inventors have developed a new skin model comprising a microbial component and a lipid component that advantageously approximates real skin. Advantageously, the cost associated with such a model is notably reduced compared to expiants. Furthermore, this model can be used with various stressors to mimic skin disorders. Indeed, the inventors have observed that applying exogenous stress to the skin model induces molecular and metabolic characteristics similar to those observed in vivo in subjects with skin exposed to sunlight or inflammatory conditions. The model can advantageously serve as a tool for screening active ingredients or cosmetic or pharmacological formulations of interest. Description of the invention

[0013] The first object of the invention therefore relates to a model of reconstituted skin comprising:

[0014] - a skin substitute, and

[0015] - a microbial component and a lipid component on the surface of said skin substitute.

[0016] Another object of the invention relates to a method for obtaining a reconstituted skin model comprising:

[0017] a. Supply of a skin substitute, b. Application to said skin substitute, concomitantly or sequentially, of a microbial component and a lipid component.

[0018] The invention also relates to a screening method for the in vitro identification of an active ingredient or cosmetic formulation to prevent and / or improve at least one skin disorder induced following at least one exogenous stress, comprising the following steps:

[0019] a. The application of said active ingredient or of said cosmetic formulation on the reconstituted skin model according to the invention; b. The application of at least one exogenous stress to said model; c. At least one measure of the expression level of at least one marker biological; and d. Determine whether said active ingredient or cosmetic formulation prevents and / or improves at least one skin disorder based on at least one level of expression measured in step c).

[0020] The invention also relates to a method for evaluating the in vitro efficacy of an active ingredient or cosmetic formulation to prevent and / or treat at least one skin disorder induced following at least one exogenous stressor including the following stages:

[0021] a. The application of said active ingredient or of said cosmetic formulation on the reconstituted skin model according to the invention; b. The application of at least one exogenous stress to said model; c. At least one measure of the expression level of at least one marker biological; and d. Evaluate the efficacy of said active ingredient or cosmetic formulation according to at least one level of expression measured in step c).

[0022] The invention also relates to an in vitro method for evaluating the tolerance of an active ingredient or cosmetic formulation, comprising the following steps:

[0023] a. The application of said active ingredient or of said cosmetic formulation on the reconstituted skin model according to the invention; b. At least one measurement of the expression level of at least one biological marker in the skin model from step a); and c. Evaluate whether the said active ingredient or cosmetic formulation is well tolerated by the skin model.

[0024] The invention also relates to a kit comprising a microbial component and a lipid component intended to be applied to a skin substitute, the microbial component comprising at least four genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium. (See Figures for description)

[0025] [Fig. 1] Chromatographic profile of collected sebum Detailed description of the invention

[0026] As indicated above, the invention relates to a reconstituted skin model comprising:

[0027] - a skin substitute, and

[0028] - a microbial component and a lipid component on the surface of said skin substitute.

[0029] By "skin substitute" is meant a skin cell culture composed of at least one layer. Skin substitutes according to the invention include, in particular, monolayer skin cell cultures, bilayer skin cell cultures, and tissue models, including reconstructed epidermis or reconstructed skin cultures. Indeed, since it is often difficult to work with fresh expiants, it is particularly advantageous, within the scope of the present invention, to use skin cell cultures. By "reconstructed epidermis" is meant an epidermis generated in vitro using conventional techniques well known to those skilled in the art (see, for example, Limât and Hunziger, Cells Tissues Organs 2002;172:79-85; Poumay et al., Arch Dermatol Res. 2004;296(5):203-11). "Reconstructed skin" refers to a culture containing at least two compartments: a dermal compartment and an epidermal compartment. Preferably, the epidermal component of the reconstructed skin contains keratinocytes. Preferably, the dermal component contains fibroblasts.

[0030] Advantageously, skin cells include normal, healthy, or pathological cells that can cause skin disorders, or cells derived from cell lines. Cultured skin cells can, in particular, be obtained from skin tissue explants. The term "explant" or "skin explant" here refers to a sample of skin cells or tissue, which may be taken for surgical purposes or for analysis. In particular, an explant can be obtained during surgical excision. "Excision" here refers to a surgical procedure consisting of cutting (excising) a more or less wide or deep portion of the skin to treat an abnormality or growth. Excision is performed either to remove a cancerous or suspected cancerous tumor, or to treat a benign skin abnormality that is bothersome, whether for functional or aesthetic reasons.An excision within the meaning of the invention includes, for example, skin samples obtained after plastic surgery (breast surgery, abdominal surgery, facelift, preputial removal, otoplasty, i.e. ear pinning, syndactyly or supernumerary finger, etc.).

[0031] A specimen can also be obtained by biopsy. Here, "biopsy" means the removal of cells or skin tissue for the purpose of analysis. Several types of biopsy procedures are known and practiced in this field. The most common types include (1) incisional biopsy, in which only a tissue sample is taken, (2) excisional biopsy (or surgical biopsy), which consists of the complete removal of a tumor mass, thus performing a therapeutic and diagnostic procedure, and (3) needle biopsy, in which a tissue sample is taken with a needle, which may be either wide or thin. Other types of biopsy exist, such as smears or curettage, and are also encompassed by the present invention.

[0032] Alternatively, said skin cells can be obtained by differentiation of stem cells (Guenou et al., Lancet, 374(9703): 1745-1753, 2009; Nissan et al., Proc. Natl. Acad. Sci., 108(36): 14861-14866, 2011; Kraehenbuehl et al., Nature Methods, 8: 731-736, 2011). Said stem cells are not human embryonic stem cells.

[0033] Skin cells according to the invention, whether they come from a biopsy or Whether obtained by stem cell differentiation, these cells comprise at least one cell type normally present in the hypodermis, dermis, and / or epidermis. These cells include, among others, keratinocytes, melanocytes, fibroblasts, adipocytes, endothelial cells, mast cells, Langerhans cells, and / or Merkel cells. Preferably, the skin cells according to the invention comprise at least keratinocytes and / or fibroblasts. More preferably, the skin cells according to the invention comprise keratinocytes and / or fibroblasts.

[0034] Many methods for culturing skin cells are known to those skilled in the art. Any of these methods can be used to culture the skin substitute of the invention. The skin substitute is cultured and / or stored under conditions that maintain, at least partially, cellular metabolism and / or cellular functions. Culture of the skin substitute therefore includes both monolayer skin cell cultures (e.g., keratinocytes) and bilayer skin cell cultures, as well as tissue models, including cultures of reconstructed skin.

[0035] Monolayer or bilayer skin cell cultures have been known and used for a very long time. Furthermore, numerous tissue models, including in particular reconstructed skin models (Rosdy et al., In Vitro Toxicol., 10(1): 39-47, 1997; Ponec et al., J Invest Dermatol., 109(3): 348-355, 1997; Ponec et al., Int J Pharm., 203(1-2): 211-225, 2000; Schmalz et al., Eur J Oral Sci., 108(5): 442-448, 2000; Black et al., Tissue Eng, 11(5-6): 723-733, 2005; Dongari-Batgtzoglou and Kashleva, Nat Protoc, 1(4): 2012-2018, 2006; Bechtoille et al., Tissue Eng, 13(11): 2667-2679, 2007; Vrana et al., Invest Ophthalmol Vis Sci, 49(12): 5325-5331, 2008; Kinicoglu et al., Biomaterials, 30(32): 6418-6425, 2009; Auxenfans et al., Eur J Dermatol, 19(2): 107-113, 2009; Kinicoglu et al., Biomaterials, 32(25): 5756-5764, 2011; Costin et al., Altem Eab Anim, 39(4): 317-337, 2011; Auxenfans et al., J Tissue Eng Regen Med, 6(7): 512-518, 2012; Lequeux et al., Skin Pharmacol Physiol, 25(1): 47-55, 2012; EP 29,678; EP 285,471; . EP 789 074; EP 1 451 302 B1; EP 1 878 790 B1; EP 1 974 718; US 2007 / 0148,771; US 2010 / 0,099,576; WO 02 / 070729; WO 2006 / 063864; WO 2006 / 0.63865; WO 2007 / 064305) are available to a person skilled in the art and are included in the scope of the invention.

[0036] Preferably, the reconstructed skin model is selected from the group comprising models of epidermis consisting mainly of keratinocytes, skin models comprising a dermis and an epidermis, and skin models comprising a dermis, an epidermis, and a hypodermis. Models comprising at least one dermis form connective tissue-like tissues, while models comprising at least one epidermis, form stratified epithelia comprising the characteristic layers of the tissue in question. For example, one can identify in epidermal models a basal layer (stratum basalis), a spinous layer (stratum spinosum), a granular layer (stratum granulosum), and a horny layer (stratum corneum).

[0037] Advantageously, said skin model is an epidermis model comprising a matrix support preferably selected from:

[0038] - an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon membrane or sponge, a semi-permeable polycarbonate or polyethylene membrane, polypropylene, polyethylene terephthalate (PET), a semi-permeable Anopore inorganic membrane, cellulose acetate or ester (HATF), a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane;

[0039] in this group we find the reconstructed Epidermis models (Skinethic®) as well as the EpiDerm® model, (Mattek Corporation);

[0040] - a film or membrane based on hyaluronic acid and / or collagen and / or fibronectin and / or fibrin.

[0041] In this group, we can particularly mention the models: Laserskin® (Fidia Advanced Biopolymers), Episkin ® (L'Oréal).

[0042] These models can be seeded by fibroblasts in the dermal part.

[0043] These models, which may or may not include fibroblasts, serve as a support for seeding keratinocytes and reconstituting the epidermis. Advantageously, in addition to keratinocytes, pigment cells, immunocompetent cells, and nerve cells are introduced; preferably, the immunocompetent cells are Langerhans cells.

[0044] There are also models dedicated to tissue therapy that can also be used within the framework of the present invention. Examples include the Epidex (Modex Thérapeutiques), Epibase® (Laboratoire Genévrier), Epicell™ (Genzyme), Autoderm™ and Transderm™ (Innogenetics) models.

[0045] The matrix support is then seeded by keratinocytes to rebuild the epidermis and ultimately obtain reconstructed skin.

[0046] Advantageously, the skin model used includes a model in which at least one complementary cell type has been incorporated, such as endothelial cells (EC) and / or immune cells such as lymphocytes, macrophages, mast cells, dendritic cells and / or adipose cells and / or skin appendages, such as hair, scalp hair, sebaceous glands.

[0047] The reconstituted skin model of the invention is particularly simple to implement. Furthermore, it does not require the use of a specific commercial cell line and is advantageously adaptable.

[0048] By "microbial component" is meant a collection of microorganisms composed of at least four different genera of bacteria. Preferably, the microbial component is a cutaneous microbial component. By "cutaneous microbial component" is meant a collection of microorganisms composed of at least four different genera of bacteria, said at least four genera of bacteria being present on the skin of a subject, preferably a human subject. Preferably, the microbial component comprises at least four, five, six, seven, eight, nine, or ten genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium, more preferably all the genera described above. Preferably, the at least four genera include the following genera: Cutibacterium, Staphylococcus, Streptococcus, and Burkholderia.The microbial component may also include one or more genera or species of fungi and / or viruses.

[0049] Preferably, the microbial component corresponds to the skin microbiota of at least one subject. "Microbiota" refers to the collection of microorganisms present in an environment. The skin microbiota therefore corresponds to the collection of microorganisms present on the skin. Of course, the composition of the microbiota can vary depending on the skin site. Preferably, the cutaneous microbial component corresponds to the skin microbiota of the face, preferably the forehead, of at least one subject. Also, according to a preferred embodiment, the cutaneous microbial component is obtained from a sample taken from at least one subject, more preferably from the face and even more preferably from the forehead. In a particular embodiment, the microbial component can be sampled from an area of ​​skin affected by a skin disorder such as, for example, an inflammatory dermatosis.Preferably, the microbial component has a viability of 5,000 RLU (2 x 10⁵ CFU / ml) at the time of application to the skin substitute surface. After 48 hours of incubation, the microbial component preferably has a viability of approximately 100,000 RLU / 0.66 cm² of skin substitute. Viability is determined using the Promega BacTiter ATP Lite kit.

[0050] By "lipid component" is meant a mixture of at least two, three, or four components selected from free fatty acids, triglycerides, squalene, wax and / or wax esters, and cholesterol and / or cholesterol esters. Preferably, the lipid component is a source of nutrients for at least one bacterial species present in the microbial component. Preferably, the component The lipid component helps protect the skin substitute and / or at least one bacterial species from desiccation. Preferably, the lipid component comprises a mixture of free fatty acids, triglycerides, squalene, wax and / or wax esters, and cholesterol and / or cholesterol esters. Preferably, the free fatty acids comprise C16:0, C18:0, C16:1, C18:1, and / or C18:2. Preferably, the waxes and / or wax esters comprise C16:0 / C16:0, C18:O / C18:O, C16:1 / C16:1, and / or C18:1 / C18:1. Preferably, the triglycerides comprise C16:0, C18:0, C16:1, and / or C18:1. Preferably, the lipid component comprises 30 to 50% w / w of triglycerides. Preferably, the lipid component comprises 15 to 30% w / w of free fatty acids. Preferably, the lipid component comprises 25 to 30% w / w of waxes and / or esters of wax. Preferably, the lipid component comprises 12 to 20% w / w of squalene. Preferably, the lipid component comprises 4.5 to 8.5% w / w of cholesterol and / or cholesterol esters. More preferably, the lipid component is composed of 30 to 50% w / w of triglycerides, 15 to 30% w / w of free fatty acids, 25 to 30% w / w of waxes and / or wax esters, 12 to 20% w / w of squalene, and 4.5 to 8.5% w / w of cholesterol and / or cholesterol esters. Preferably, the lipid component comprises 3.0 to 6.0% w / w of cholesterol and 1.5 to 2.5% w / w of cholesterol esters. Preferably, the lipid component is in hydrolipidic form, i.e. in the form of an emulsion with water. Preferably, the lipid component is sebum. Preferably, the lipid component is taken from a sample of at least one subject, more preferably from the face and even more preferably from the forehead.Preferably, the lipid component is added at a final concentration of 30 to 70 pg / cm2, more preferably at a final concentration of 40 pg / cm2 of skin substitute. Since the skin substitute is composed of at least one layer of skin cells, both the microbial and lipid components are present on the surface of said substitute.

[0051] The term "subject" herein means any human being, whether adult or child. According to the invention, "child" means an individual aged 16 years or younger. Thus, the category of children according to the invention includes newborns aged between 0 and 1 month, infants aged between 1 month and 2 years, and children properly speaking, who are at least 2 years old. A "newborn," as understood herein, may be born at term or prematurely. An "adult" within the meaning of the present invention is a person who is not a child, in other words, a person over the age of 16.

[0052] Another object of the invention relates to a method for obtaining a reconstituted skin model comprising:

[0053] a. Supply of a skin substitute, b. Application to said skin substitute, either concomitantly or sequentially, of a microbial component and a lipid component.

[0054] The skin substitute is as described above. The lipid component can be applied before the microbial component, or vice versa. Preferably, the microbial and lipid components are mixed together before application to the skin substitute. Preferably, the microbial and / or lipid components are applied to the skin substitute using a pipette and a finger cot. Preferably, the microbial component is applied fresh; that is, it has not undergone a prior freezing step.

[0055] According to one particular aspect, the reconstituted skin is stressed. Preferably, the method of obtaining it further comprises a step of carrying out at least one exogenous stress. Also, according to another aspect, the method of obtaining a reconstituted skin model is characterized in that the reconstituted skin is stressed and in that the method further comprises a step

[0056] c) of the occurrence of at least one exogenous stress.

[0057] Step c) can be performed before or after step b) of the process for obtaining the model. In some cases, step c) can be performed on the microbial component and / or the lipid component before their application to the skin substitute. Preferably, step c) is performed after step b) of the process.

[0058] “Exogenous stress” means any external factor affecting the integrity of the skin and potentially leading to a progressive decrease in the effectiveness of its functions. Exogenous stress can be chemical and / or physical.

[0059] Physical stresses include, in particular, environmental stresses (e.g., temperature) and mechanical stresses (e.g., friction). Preferably, the physical stress is chosen from ultraviolet radiation, sunlight, infrared, near-infrared, thermal, radiofrequency radiation including microwaves and mobile phone waves, ionizing radiation including beta, gamma, X-rays, non-ionizing radiation, radiation from a magnetic field, ozone, a change in pressure, heat, cold, friction, stretching, particles.

[0060] Preferably, the physical stress is radiation mimicking solar radiation, more preferably irradiation in the UV range, including UVA and / or UVB; and / or visible light, including high-energy blue visible light and / or infrared. "Ultraviolet radiation" means electromagnetic rays with wavelengths from 153 nm to 400 nm. Preferably, UV exposure according to the invention includes exposure to UVA and / or UVB. For the purposes of this invention, UVA means radiation with wavelengths from 400 to 320 nm. For the purposes of this invention, UVB means radiation with wavelengths from 320 to 290 nm. "Visible light" means radiation with wavelengths from 400 to 700 nm. "High-energy blue visible light" refers to visible light with a wavelength between 400 and 450 nm. "Infrared" refers to radiation with a wavelength between 700 nm and 2500 nm.

[0061] According to a preferred embodiment, UV exposure according to the invention comprises or consists of exposure to UVA. According to another preferred embodiment, UV exposure according to the invention comprises or consists of exposure to UVB. According to an advantageous embodiment, UV exposure according to the invention comprises or consists of exposure to both UVA and UVB. According to yet another advantageous embodiment, radiation exposure according to the invention comprises or consists of exposure to UVA and / or UVB and / or high-energy blue visible light and / or IR. In one particular embodiment, the applied wavelengths are between 290 and 450 nm or between 290 and 400 nm. In another particular embodiment, the skin model is exposed to a single acute UV dose of 16.5 J / cm² for approximately 45 minutes.In another specific embodiment, the UVB dose is between 80 and 150 mJ / cm² of UVB; preferably 120 mJ / cm² of UVB. Irradiation can be carried out under conditions well known to those skilled in the art (see, for example, Lordanov et al., J. Biol. Chem. 1998).

[0062] Preferably, the chemical stressor is at least one chemical substance whose topical application leads to an alteration in the structure and / or function of the skin. Preferably, the chemical stressor is at least one allergen, a pollutant, a surfactant, a solvent, or at least one pro-inflammatory stimulating reagent such as a cytokine; preferably a cocktail of specific cytokines capable of inducing the skin disorder under investigation. Such reagents are well known and are described, for example, in application WO 2015 / 014949. According to a particular aspect, the chemical stressor comprises one or two components selected from interleukin 1b, a TLR-2 receptor ligand, and a TLR-3 receptor ligand. Preferably, the chemical stressor comprises interleukin 1b and optionally the TLR ligand (Poly (LC) and / or Pam3CSK4), more preferably all three components.Inducing chemical stress, including the application of at least interleukin IL-1 [> to the reconstructed skin model, advantageously mimics atopic dermatitis (AD). Of course, other chemical stresses can also mimic AD, such as the combination of Poly I:C and TNFα; the combination of IL-4 and IL-13; the combination of Poly I:C, IL-4, IL-13; the combination of IL-4, IL-13, IL-31 and optionally TNFα; and the combination of IL-4, IL-13, IL-22, and TNFα.

[0063] By way of specific example, in order to mimic the effect of pollution on the skin, particles or chemical molecules may be brought into contact with the skin substitute. For example, "particle matter" (PM 10), from ERM- batches CZ100 / CZ120 are in the form of a very fine powder. For example, 10 pL / cm² of acetone-based solution of these fine particles can be deposited on the surface of reconstructed epidermis before the addition of microbiota and sebum. Alternatively, benzo(a)pyrene (B(a)P) can be applied to the surface of the skin substitute. B(a)P is a good example of an outdoor pollutant because it leads to highly toxic metabolites. Alternatively, tetrabisphenol A (TBBPA), a brominated flame retardant found in most household furnishings (furniture, fabrics, etc.) and considered an indoor pollutant, can be applied to the surface of the skin substitute. Finally, cortisol is known as the stress hormone. High and persistent levels of cortisol can be very damaging to the skin.Negative effects may include undesirable visible signs of aging such as wrinkles and fine lines, skin thinning, reduced elasticity, and decreased skin barrier functionality. To obtain a model of stressed skin, step (c) may involve exposing the skin substitute to cortisol, for example, at a concentration of 4 pM.

[0064] Without being limiting, certain exogenous stresses, allowing to mimic certain skin disorders, which can be carried out in the context of the present invention, as well as the markers associated with said skin disorder which can be measured in the context of the present invention are described in Table 1 below.

[0065] [Tables 1] Disorder / Pathology Stress Associated Markers DA IL4+IL13+IL31 mixture ±TNFa, mixture (ILβ + Poly(I:C) + Pam3CSK4), mixture IL4+IL13+IL22+TNFa TSLP, IL8, JAK / STAT pathway, fi-laggrin, Claudin 1, antimicrobial peptides Acne biofilm or Cutibacterium acnes membrane extract; squalene peroxidase antimicrobial peptides, IL8, IL6, IL17, lipogenesis inhibition Psoriasis Cocktail IL17+OSM+TNFa ± IMQ(imiquimod); activated TH17 ± IMQ IL8, IL6, PAMs, Ki67 Rosacea FSL1+LL37+TNFa IL8, VEGF, IL6, MMP9 Pruritus IL31; TSLP; NGF Substance P, CGRP, Histamine, SEMA3A, JAK / STAT Dry skin Xerosis Dehydration FNH, epidermal proteases, caspase 14, peptidyl arginine desiminase, fi-laggrin, ceramides Reactive skin SDS, Histamine Substance P, CGRP;Histamine; Alteration of barrier function; SDS, Stripping; Claudine, Filaggrin, Transglutaminase-1, desmoglein, ceramides, fatty acids, lipid barrier organization; Aging; UVA, tobacco smoke, blue light, cortisol, ozone, urban dust; Oxidative stress, glutathione, extracellular matrix (collagen, hyaluronic acid, glycosaminoglycan, elastin fibers), metalloproteinases (e.g., MMPI), melanin; Pollution; Ozone, particles, PAHs, heavy metals, TBBPA; Passage and metabolism of PAHs, metabolic enzymes (CYP1A1, CYP1B1...), transporters, oxidative stress, MD A, lipids, MMPs, melanin, collagen, oxidation of; Proteins, inflammation... Solar irradiation (UV, UVB, UVA), blue light, infrared radiation, apoptosis (caspase-3, sunburn cells), viability (MTT test, LDH), genotoxicity (DNA lesions, CPD 64PP), inflammation (IL8, IL6, prostaglandins), oxidative stress (malondialdehyde, catalase, superoxide dismutase, qPCR), extracellular matrix degradation and proteases (MMPI, elastase, collagen, elastin, GAG), endogenous stress (cortisol, stratum corneum lipids, epidermal thickness), stretch marks, keratinocyte migration, fibroblast proliferation, MMP9

[0066] The invention also relates to a model of reconstituted skin obtained by the process as described herein.

[0067] In a particular embodiment, at least two exogenous stresses, identical or different, are performed on the reconstituted skin model according to the invention.

[0068] In another aspect, the invention makes it possible to identify active ingredients or formulations for preventing and / or improving at least one skin condition, or to evaluate the efficacy of active ingredients or formulations for preventing and / or improving at least one skin condition. In particular, the invention makes it possible to distinguish active ingredients or formulations according to their activity in preventing and / or improving at least one skin condition.

[0069] The invention therefore also relates to a screening method for the in vitro identification of an active ingredient or cosmetic formulation to prevent and / or improve at least one skin disorder induced following at least one exogenous stress, comprising the following steps:

[0070] a. The application of said active ingredient or of said cosmetic formulation on the reconstituted skin model; b. The application of at least one exogenous stress to said model; c. At least one measure of the expression level of at least one marker biological; and d. Determine whether said active ingredient or cosmetic formulation prevents and / or improves at least one skin disorder based on at least one level of expression measured in step c).

[0071] The invention also relates to a method for evaluating the in vitro efficacy of an active ingredient or cosmetic formulation to prevent and / or treat at least one skin disorder induced following at least one exogenous stress, comprising the following steps:

[0072] a. The application of said active ingredient or said cosmetic formulation to the reconstituted skin model; b. The application of at least one exogenous stress to said model; c. At least one measure of the expression level of at least one marker biological; and d. Evaluate the efficacy of said active ingredient or cosmetic formulation according to at least one level of expression measured in step c).

[0073] Step a) can be performed before or after step b) of the screening method for the in vitro identification of an active ingredient or cosmetic formulation, or in the method for evaluating the in vitro efficacy of an active ingredient or cosmetic formulation. Indeed, performing step a) before step b) can, in particular, allow for the evaluation of the ability of an active ingredient or cosmetic formulation to prevent at least one skin condition, whereas performing step a) after step b) can, in particular, allow for the evaluation of the ability of an active ingredient or cosmetic formulation to improve at least one skin condition.

[0074] The application of the active ingredient of interest to the skin model according to step a) can be carried out directly. Alternatively, it may be advantageous to formulate the active ingredient of interest, for example, to obtain a liquid composition, in order to facilitate its application to the skin model. Thus, according to one embodiment of the invention, the process further comprises a step of formulating the active ingredient, in particular in the form of a liquid solution, especially an aqueous one, prior to step a) of applying said active ingredient to a skin model.

[0075] The candidate active ingredient is an active ingredient for the prevention and / or improvement of at least one skin disorder if said candidate active ingredient allows for the modulation of the expression of at least one biological marker of the invention. This modulation may correspond, depending on the case, and in particular depending on the nature of the biological marker, to an increase or a decrease in the expression of said marker. Similarly, the candidate formulation is a formulation for the prevention and / or improvement of at least one skin disorder if said candidate formulation allows for the modulation of the expression of at least one biological marker of the invention. This modulation may correspond, depending on the case, and in particular depending on the nature of the biological marker, to an increase in the expression of said marker. mentation or a decrease in the expression of said marker.

[0076] For example, it would be advantageous to identify active ingredients or formulations that minimize the effects of exogenous stress or skin disorder on barrier markers, in order to maintain the integrity of the skin barrier.

[0077] For the purposes of this application, "the in vitro efficacy of an active ingredient or cosmetic formulation to prevent and / or treat at least one skin disorder" means the ability of the formulation or active ingredient to reverse or reduce the effects associated with at least one skin disorder. Prevention in this case refers to treatment administered before the development of the skin disorder (i.e., before the occurrence of exogenous stress in step b)), while reduction refers to treatment administered once the effects of the skin disorder have appeared (i.e., after the occurrence of exogenous stress in step b)).

[0078] The term "augmented", as used here, means a larger quantity, for example, a quantity slightly greater than the original quantity, or for example a quantity in large excess compared to the original quantity, and in particular all quantities in the interval.Alternatively, "increase" may refer to a quantity or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% greater than the quantity or activity to which the increased quantity or activity is being compared, or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, or 650%. 700%, 750%, 800%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900% or 2000% more than the quantity or activity to which the increased quantity or activity is being compared. The terms "increased", "greater than", and "increased" are used interchangeably here.

[0079] The term "reduced", as used here, means a smaller quantity, for example, a quantity slightly less than the original quantity, or for example a quantity greatly reduced compared to the original quantity, and in particular all quantities in the interval. Alternatively, "decrease" may refer to a quantity or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% less than the quantity or activity to which the decreased quantity or activity is being compared, or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, or 650%. %, 700%, 750%, 800%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900% or 2000% less than the quantity or activity for. to which the decreased quantity or activity is compared. The terms "decreased", "less than", and "reduced" are used interchangeably here.

[0080] In order to determine whether the expression of a marker is increased or decreased in the skin model of the invention, the level of expression of said marker may be compared with a reference level of expression.

[0081] According to this preferred embodiment, step d), which determines whether the active ingredient or cosmetic formulation prevents and / or improves at least one skin disorder, may include a comparison of the expression level of the biological marker from step c) with a reference expression level.

[0082] For the purposes of this application, "a reference expression level of a biological marker" means any expression level of that marker used as a reference. For example, a reference expression level may be obtained by measuring the expression level of the marker of interest in a normal skin model. Such a normal skin model may, for example, be a reconstructed skin model obtained from skin cells of a healthy subject or one without apparent pathology. A reconstructed skin model without the application of exogenous stress may also be used as a normal skin model. In this case, the expression of the biological marker may be measured before step b); the level thus determined is then the reference expression level of that biological marker.

[0083] In other embodiments, the reference expression level of a biological marker corresponds to the expression level of said marker in the skin model in the absence or presence of a particular treatment. For example, in one particular embodiment, the reference expression level of a biological marker is obtained by measuring the expression of said marker in the skin model that has not been exposed to an active ingredient or formulation and / or that has not been subjected to exogenous stress. In another particular embodiment, the expression of said marker is measured in the skin model treated with an active ingredient or formulation known to be effective against a skin disorder.

[0084] A person skilled in the art will also readily understand that the comparison in step (d) is preferably made between measurements of expression levels obtained for skin models obtained from skin substitutes of similar, or even identical, histological structures. For the purposes of this application, "similar histological structures" means that the relative proportions of the cell types included in the compared skin models are similar. Thus, it is preferable that the relative proportions of the cell types and / or microorganism types included in the skin model in step (a) do not differ by more than 5% from the relative proportions of the cell types and / or microorganism types included in the skin model used to obtain the reference expression level in step (a). (d) For the purposes of this application, "relative proportion of a cell type" means the ratio of the number of cells of that cell type to the total number of cells in the skin model. For example, it is preferable that the proportion of keratinocytes to the total number of cells in the skin model in step (a) not differ by more than 5% from the proportion of keratinocytes to the total number of cells in the skin model used to obtain the reference expression level in step (d). For the purposes of this application, "relative proportion of a type of microorganism" means the ratio of the abundance of a genus or species of microorganism to the total number of microorganisms in the skin model. Preferably, the relative proportion of a genus or species of bacteria is the ratio of the abundance of that genus or species to the total number of bacteria.

[0085] For the purposes of this application, "identical histological structures" means that the relative proportions of the cell types included in the compared skin models are identical. For the purposes of this invention, the relative proportions of the cell types included in the skin model of step a) are identical to the relative proportions of the cell types included in the skin model used to obtain the reference expression level of step d) when they do not differ by more than 0.1%. Advantageously, the proportion of keratinocytes to the total number of cells in the skin model of step a) does not differ by more than 0.1% from the proportion of keratinocytes to the total number of cells in the skin model used to obtain the reference expression level of step d).

[0086] A person skilled in the art will also readily understand that the comparison in step d) is preferably made between measurements of expression levels obtained for skin models that are of similar, or even identical, size, volume, or weight. Thus, it is preferable that the size, volume, or weight of the skin model in step a) not differ by more than 5% from the size, volume, or weight of the skin model used to obtain the reference expression level in step d). More preferably, the size, volume, and weight of the skin model in step a) not differ by more than 5% from the size, volume, and weight of the skin model used to obtain the reference expression level in step d).Even more preferably, the size, volume and weight of the skin model in step a) do not differ by more than 0.1% from the size, volume and weight of the skin model used to obtain the reference expression level in step d).

[0087] Alternatively, if the skin models differ by more than 5% in size, volume and weight, a person skilled in the art can normalize the level obtained in step c) and the reference level in step d) using a normalization factor.

[0088] This normalization factor could, for example, be a directly accessible physical marker such as the mass of cells in the sample, or the mass of a cellular constituent, such as the mass of cellular DNA or the mass of cellular proteins.

[0089] It may also be advantageous to use as a normalization factor the expression level of a gene that is expressed at the same level in all, or nearly all, cells of the organism. In other words, according to a particular embodiment of the present invention, the expression level of a housekeeping gene is used as the normalization factor. According to another embodiment, the level obtained in step c) and the reference level in step d) are normalized using the expression level, not of the housekeeping genes, but of the proteins encoded by them. A housekeeping gene is a gene expressed in all cell types that encodes a protein having a basic function necessary for the survival of all cell types. A list of human housekeeping genes can be found in Eisenberg et al. (Trends in Genet, 19: 362-365, 2003).The housekeeping genes according to the invention include, for example, the genes RPS28, GAPDH, B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1, HPRT1, IPO8 and HMBS.

[0090] Step c) is performed after steps a) and b). The determination in step d) is advantageously carried out by comparing the expression level in step c) with a reference expression level (see above). For example, a skin model that has not been treated with the active ingredient or formulation can be used as a control. In this case, the expression level of the biological marker of the invention is measured in said skin model before and after application of the active ingredient or cosmetic formulation. Alternatively, the expression level of the biological marker of the invention is measured in a skin model without application of the active ingredient or cosmetic formulation and in a skin model with the active ingredient or cosmetic formulation. The expression level of the biological marker of the invention can also be compared with that measured in vivo in normal skin or in vivo in skin subjected to exogenous stress.By "normal skin," we mean skin from a healthy individual or one without any apparent skin pathology.

[0091] The reference expression level is preferably the expression level of said biological marker in a skin model that has not been in contact with the active ingredient or formulation, which allows for a meaningful comparison between the expression level in step c) and said reference level. For example, a skin model that has not been treated with the active ingredient or formulation (i.e., without performing step a) or before performing step a)) can be used as a control. In this case, the expression level of the biological marker of the invention is measured in said skin model with and without application of the active agent or cosmetic formulation (or before and after completion of step a)).

[0092] The method of the invention may further include a comparison of cell viability in the skin model treated with the active ingredient or formulation and in the control sample. In this case, the active ingredient or cosmetic formulation is well tolerated by the skin if the cell viability of the sample is not affected by the presence of the active ingredient or cosmetic formulation.

[0093] According to another preferred embodiment, the process of the invention therefore includes an additional step of determining cell viability in the skin model treated with the active ingredient or cosmetic formulation, determining cell viability in the control sample and comparing the two.

[0094] Numerous tests for determining cell viability are available to those skilled in the art and are commonly used in cosmetology. In particular, the MTT test, described for example in Mosman et al. (J Immunol Methods, 65(1-2): 55-63, 1983), will be mentioned.

[0095] The term “skin disorders” herein refers to all abnormal reactions that may occur on an individual’s skin. These conditions affect the skin itself (i.e., the epidermis, dermis and / or hypodermis), as well as the skin pores, the sweat and sebaceous glands associated with it, and / or the microbiota.

[0096] The skin disorders according to the invention are characterized more particularly by lesions, which correspond to damaged or unhealthy skin. Damaged skin includes, for example, sensitive reactive skin, dry skin (xerosis), skin damaged by the sun, radiation, cold, stress or pollution, by an allergy, by urticaria, by eczema and other forms of dermatitis such as atopic dermatitis, impetigo, irritant dermatitis, in particular irritant dermatitis of the diaper area or diaper rash, contact dermatitis, seborrheic dermatitis of the skin and scalp (cradle cap), psoriasis, Lainer-Moussous disease, or even by wounds or burns.Skin disorders encompass a wide range of conditions, including eczema, angiomas (including tuberous, subcutaneous, and flat angiomas), hemangiomas, infant acne, adolescent acne, ichthyosis (e.g., ichthyosis vulgaris, congenital ichthyosis, harlequin ichthyosis), rosacea, pruritus, and more. A skin disorder can also be caused or exacerbated by an external infection, such as one of parasitic, viral, bacterial, or fungal origin. Skin disorders include, in particular, warts, prurigo strophulus, scabies, head lice, and mycoses. Mycoses are parasitic infections caused by the proliferation of microscopic fungi that parasitize the body. Among the most common mycoses are candidiasis and pityrosporum, which are caused by the overgrowth of yeasts. the skin. Preferably, the skin disorder is chosen from among disorders related to aging, pollution, radiation; inflammatory dermatoses, preferably atopic dermatitis, acne, psoriasis; rosacea; pruritus; an alteration of the barrier function; reactive skin; xerosis.

[0097] For the purposes of this application, a "biological marker" means a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biological marker thus encompasses a wide range of substances and parameters. For example, a biological marker may be a substance whose detection indicates a particular pathological state (e.g., the presence of C-reactive protein as a marker of infection), or conversely, a substance whose detection indicates a specific physiological state. Preferably, the biological marker is a biological marker of a skin disorder if its level of expression differs in healthy skin and in skin exhibiting the clinical characteristics of that skin disorder.More preferably, the said marker characterizes a skin disorder if the said marker is differentially expressed in a skin model in which exogenous stress has been induced and the skin model not subjected to exogenous stress. This results in a ratio in step d) that is different from 1 when the measurement of the expression level is compared between these two conditions.

[0098] The biological marker according to the invention is preferably a gene, the products of a gene such as its transcripts and peptides derived from its transcripts, a lipid, a sugar, or a metabolite. Examples of biological markers according to the invention are provided in particular in Table 1, as well as below. For each of these types of markers, numerous methods are available to those skilled in the art for measuring the expression of said biological marker.

[0099] In a particular embodiment, at least two, three, four, five, six, seven, eight, nine, or ten biomarkers are measured. These biomarkers are discriminating, thus making it possible to screen cosmetic active ingredients or formulations for preventing or treating skin disorders or to evaluate the efficacy of cosmetic active ingredients or formulations for preventing or treating skin disorders.

[0100] A person skilled in the art seeking to determine which class a biological marker belongs to can easily consult the relevant scientific literature or refer to public databases such as, for example, those grouped on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0101] The biological marker of the invention is advantageously a marker selected from among the markers of inflammation, oxidative stress, barrier function, and / or the microbial component. The biological marker can be measured in the skin substitute and / or in the microbial / lipid component present on the surface of said skin substitute in the model. According to one approach, the biological marker is measured at least in the microbial component, preferably in the microbial component. According to another approach, the biological marker is measured at least in the skin substitute, preferably in the skin substitute.

[0102] The "inflammation markers" according to the invention include markers that are modulated during an inflammatory response in the skin. They include cytokines, such as IL-8 or TSLP, as well as antimicrobial peptides, such as psoriasin, encoded by the S100A7 gene, and beta-defensin 2, encoded by the DEFB4A gene.

[0103] Among cytokines, IL-8 is a cytokine that is induced early in skin inflammation. It is notably produced by de novo synthesis in keratinocytes stimulated by IL-1 and / or TNF. Therefore, it is a predictive marker of a biological response, even in the absence of visible signs of skin irritation. The protein sequence of human interleukin IL-8 corresponds to the NCBI reference sequence: NP_000575. This protein is encoded by the human IL8 gene (NCBI reference: Gene ID: 3576). The sequence of this gene is available under NCBI reference: NM_000584.

[0104] The TSLP protein is a characteristic inflammatory cytokine of atopic dermatitis (AD). Its secretion is markedly stimulated in atopic dermatitis and promotes the initiation of an inflammatory response by Th2 cells. Therefore, it is a good marker of AD. Other markers, such as RANTES / CCL5, MCP-3 / CCL7, and / or IL-6, are also indicative of AD and can be measured within the scope of the present invention.

[0105] The "oxidative stress markers" according to the invention include markers that are expressed in the skin and / or in the microbial component under oxidative stress. Preferably, the oxidative stress markers are metabolites. Preferably, at least one metabolite among xanthine, uric acid, and glutathione is measured.

[0106] The "barrier function markers" according to the invention comprise markers that are specifically expressed in the outermost layers of the epidermis and that participate in the barrier function. Preferably, the barrier function markers according to the invention are markers expressed in the stratum comeum or markers expressed in the tight junctions of the stratum granulosum. These markers include, in particular, claudins, including Claudin-1 (CLDN1), transglutaminases, such as transglutaminase 1 (TGM1), the Keratins, including keratin 1 (KRT1) and keratin 10 (KRT10), human peptidyl arginine deiminase type 1 (PAD1), caspase 14 (CASP14), aquaporin 3 (AQP3), loricin (LOR), sciellin (SCEL), BARX Homeobox 2 protein (BARX2), desmoglein-1 (DSG1), filaggrin (FLG), involucrin (IVL), sphingomyelin diesterase (SMPD), comeodesmosin (CSDN), etc. Preferably, at least one marker among Claudin-1 (CLDN1), transglutaminase 1 (TGM1), and filaggrin (FLG) is measured. In another aspect, barrier function markers include one or more discriminating metabolites of barrier function. Preferably, at least one metabolite among octanoic acid, serine, glutamate, phosphorylcholine, histidine, L-glutamine, L-alanine, methionine, and xanthine is measured.

[0107] The "markers of the microbial component" according to the invention include markers that are modulated in the microbial component in response to exogenous stress and / or a cosmetic active ingredient or formulation. They include, in particular, metabolites such as formate, acetate, glucose and maltose, glycerol, betaine, octanoate, tyrosine, panthenol, and phenylalanine.

[0108] Gene expression can be measured, for example, at the nucleotide level, by measuring the quantity of transcripts of said gene, and can also be measured, for example, at the peptide level, by measuring, for example, the quantity of proteins derived from said transcripts. Thus, "measuring the level of gene expression" means, for the purposes of this invention, measuring the quantity or cellular concentration of the gene product in its peptide or nucleotide form. In particular, the expression of at least one gene (e.g., 16S) can be measured in order to determine the presence or absence of at least one microorganism in the model microbiota.

[0109] In general, the expression of the biological marker according to the invention will be detected in vitro from the reconstituted skin model.

[0110] In a particular embodiment, the method of the invention may include one or more intermediate steps between obtaining the reconstituted skin model and measuring the expression of the biological marker, said steps corresponding to the extraction from said reconstituted skin model of a lipid sample, an NMF sample, an mRNA sample (or the corresponding cDNA), or a protein sample. This can then be used directly to measure the expression of the marker. The preparation or extraction of mRNA (as well as its reverse transcription into cDNA), proteins, lipids, or NMF from a skin cell sample are merely routine procedures well known to those skilled in the art.

[0111] In the case of markers that are secreted into the culture medium, a person skilled in the art can simply dose the marker from said culture medium.

[0112] Once a sample of mRNA (or corresponding cDNA) or protein is obtained, the expression of the marker can be measured either in the mRNAs (i.e., in all the mRNAs or cDNAs present in the sample) or in the proteins (i.e., in all the proteins present in the sample). The method used for this then depends on the type of transformation (mRNA, cDNA, or protein) and the type of sample available.

[0113] When the expression of the marker is measured at the level of the corresponding mRNA (or cDNA), any technology commonly used by a person skilled in the art can be implemented. These gene expression level analysis technologies, such as transcriptome analysis, include well-known methods such as PCR (Polymerase Chain Reaction, if starting from DNA), RT-PCR (Reverse Transcription-PCR, if starting from RNA) or quantitative RT-PCR, or even nucleic acid microarrays (including DNA microarrays and oligonucleotide microarrays) for higher throughput.

[0114] The term “nucleic acid chips” herein means several different nucleic acid probes attached to a substrate, which may be a microchip, a glass slide, or a microsphere-sized bead. The microchip may be made of polymers, plastics, resins, polysaccharides, silica or a silica-based material, carbon, metals, inorganic glass, or nitrocellulose.

[0115] The probes may be nucleic acids such as cDNAs (“cDNA chip”), mRNAs (“mRNA chip”) or oligonucleotides (“oligonucleotide chip”), said oligonucleotides typically having a length of between about 25 and 60 nucleotides.

[0116] To determine the expression profile of a particular gene, a nucleic acid corresponding to all or part of said gene is labeled and then brought into contact with the chip under hybridization conditions, leading to the formation of complexes between said labeled target nucleic acid and probes attached to the surface of the chip that are complementary to this nucleic acid. The presence of labeled hybridized complexes is then detected.

[0117] These technologies make it possible to monitor the expression level of a particular gene, several genes, or even all the genes in the genome (full genome or full transcriptome) in a biological sample (cells, tissues, etc.). These technologies are used routinely by those skilled in the art, and therefore it is not necessary to describe them in detail here. Examples of implementations of the invention based on gene expression analysis (cDNA microarrays) and quantitative PCR are described in the experimental section.

[0118] Alternatively, any current or future technology that allows gene expression to be determined on the basis of the amount of mRNA may be used in the sample. For example, a professional can measure gene expression by hybridization with a labeled nucleic acid probe, such as Northern blot (for mRNA) or Southern blot (for cDNA), but also by techniques such as Serial Analysis of Gene Expression (SAGE) and its derivatives, such as LongSAGE, SuperSAGE, DeepSAGE, etc. Tissue microarrays (also known as TMAs) can also be used. Tests commonly used with tissue microarrays include immunohistochemistry and fluorescence in situ hybridization (FISH). For mRNA analysis, tissue microarrays can be combined with FISH. Finally, whole-transcriptome shotgun sequencing (RNA-Seq) can be used to determine the amount of mRNA in the sample.To this end, several methods of massive parallel sequencing are available. Such methods are described in, for example, US 4,882,127; US 4,849,077; US 7,556,922; US 6,723,513; WO 03 / 066896; . WO 2007 / 111924; US 2008 / 0020392; WO 2006 / 084132; US 2009 / 0186349; US 2009 / 0181860; US 2009 / 0181385; US 2006 / 0275782; EP-B1-1141399; Shendure & Ji, Nat Biotechnol., 26(10): 1135-45, 2008; Pihlak et al., Nat Biotechnol., 26(6): 676-684, 2008; Fuller et al., Nature Biotechnol., 27(11): 1013-1023, 2009; Mardis, Genome Med., 1(4): 40, 2009; Metzker, Nature Rev. Genet., 11(1): 31-46, 2010.

[0119] When the expression of the marker is measured at the protein level, it is possible to use specific antibodies, in particular in well-known technologies such as immunoprecipitation, immunohistochemistry, western blot, dot blot, ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled with immunohistochemistry.Other techniques that can be used include FRET or BRET techniques, flow cytometry, microscopy or histochemistry methods, including confocal microscopy, fluorescence microscopy, electron microscopy, atomic force microscopy, methods based on the use of one or more excitation wavelengths and a suitable optical method, such as an electrochemical method (voltametry and amperometry techniques), and radio frequency methods, such as multipolar, confocal and non-confocal resonance spectroscopy, such as the detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., by surface plasmon resonance, ellipsometry, resonant mirror method, etc.), radioisotope or magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); by two-dimensional electrophoresis, by spectrophotometry, by spec. Mass chromatography and tandem mass spectrometry, by liquid or gas chromatography coupled with mass spectrometry or tandem mass spectrometry. All these techniques are well known to those in the field and it is not necessary to detail them here.

[0120] If the biological marker is a lipid, in particular a ceramide or a fatty acid, or a metabolite, a person skilled in the art may use any available methods to measure the lipid content in a sample of skin cells. These methods include, among others, high-performance liquid chromatography (HPLC, see for example Sullivan et al., Arch Ophthalmol., 120(12): 1689-99, 2002), high-performance liquid chromatography coupled with evaporative light diffraction (HPLC-ESD, see Nordback et al., J. High Resolut. Chromatogr., 22: 483-486, 1999; Torres et al., J. Chromatogr. A., 1078: 28-34, 2005); thin-layer chromatography (TLC, e.g. Downing et al., J Invest Dermatol., 77(4): 358-360, 1981; Nordstrom et al., J Invest Dermatol., 87(2): 260-263, 1986); nuclear magnetic resonance (NMR, see e.g. Robosky et al., J Lipid Res., 49(3): 686-692, 2008); in vivo confocal Raman microspectroscopy; mass spectrometry, gas chromatography coupled to mass spectrometry (GC-MS, see O'Neill et al., J Chromatogr Sci., 14(1): 28-36, 1976); gas chromatography coupled to a flame ionization detector; liquid chromatography coupled to mass spectrometry (see, for example, van Smeden et al., J Lipid Res, 52(6):1211-1221, 2011); ultra-high-performance liquid chromatography (UPLC, see Rainville et al., J Proteome Res., 6(2):552-558, 2007; Castro-Perez et al., J Proteome Res., 10(9): 4281-4290, 2011). The organization of these lipids in the skin, and more specifically in the stratum corneum (or horny layer), lamellar or lateral organization, can also be analyzed using techniques such as X-ray diffraction (Bouwstra et al., J Invest Dermatol., 97(6): 1005-1012, 1991; van Smeden et al., J Lipid Res., 52(6):1211-1221, 1991) or by Fourier transform infrared spectroscopy (Gorcea et al., Int J Pharm. Nov 10, 2011) or by morphometric analysis by electron microscopy (Daehnhardt-Pfeiffer et al., Skin Pharmacol Physiol., 25(3): 155-161, 2012) or by electron microscopy analysis of vitreous skin section combined with molecular analysis (Iwai et al., JInvest Dermatol., Apr 26, 2012). .

[0121] NMF measurement is a procedure well known to those skilled in the art. In particular, NMF can be measured using in vivo confocal Raman microspectroscopy. This procedure has been commonly used in the field for at least 15 years. Examples include the publications by Caspers et al. (J Invest Dermatol., 116(3): 434-442, 2001), Vyumvuhore et al. (J Biomed Opt., 19(11): 111603, 2014), and Falcone et al. (Skin Pharmacol Physiol, 28: 307-317, 2015). It is also possible to quantify NMFs by liquid chromatography coupled with mass spectrometry. See, for example, Piraud et al. (Rapid Common Mass Spectrom, 19(12):1587-602, 2005), Petritis et al. (Journal of Chromatography A, 833(2):147-155, 1999), Henriksen et al. (J Am Soc Mass Spectrom, 16(4):446-455, 2005) or Yang (Application of biophysics and bioengineering to the assessment of skin barrier function. Thesis (Doctor of Philosophy (PhD)). University of Bath, UK, 2011).

[0122] In a particular embodiment, the evaluated active ingredient is at least a sunscreen. By sunscreen, we mean any substance capable of filtering solar radiation to protect a surface, typically the skin or hair, against the harmful effects of this radiation.

[0123] The solar filter is chosen from among those classically known, of which

[0124] - organic sunscreens such as triazine derivatives, benzotriazole and phenyl benzotriazole derivatives, dibenzoylmethane derivatives, benzophenone and amino-substituted 2-hydroxybenzophenone derivatives, merocyanine derivatives including methoxypropylamino cyclohexenylidene ethoxyethylcyanoacetate, para-aminobenzoic acid derivatives, salicylic derivatives, cinnamic derivatives, [3,[3'-diphenylacrylate] derivatives, benzylidene camphor derivatives, phenylbenzimidazole derivatives, anthranilic derivatives, imidazoline derivatives, and benzalmalonate derivatives. - inorganic sunscreens such as coated or uncoated metallic oxides such as titanium, iron, zinc, zirconium, or cerium oxides.

[0125] Preferably, the active ingredient evaluated is at least a sunscreen. Preferably, the method for evaluating the in vitro efficacy of at least one sunscreen or a cosmetic formulation comprising it or them for preventing and / or treating at least one skin disorder induced following at least one exogenous stress consisting of UV, infrared, or blue light irradiation.

[0126] Method for in vitro evaluation of the tolerance of an active ingredient or cosmetic formulation, comprising the following steps:

[0127] a. The application of said active ingredient or cosmetic formulation to the reconstructed skin model; b. At least one measurement of the expression level of at least one biological marker in the skin model from step a); and c. Evaluate whether the said active ingredient or cosmetic formulation is well tolerated by the skin model.

[0128] The active ingredient is an active ingredient that is well tolerated by the skin if said active ingredient does not modulate The expression of the biological marker is significant. Similarly, the cosmetic formulation is well tolerated if the expression of the biological marker is not significantly modulated by its addition to the reconstructed skin model. "Significant" means a result obtained by performing a statistical test, such as a univariate Wilcoxon test, with a p-value less than 0.05, preferably less than 0.01, and even more preferably less than 0.001. The appropriate statistical tests are well known to those skilled in the art. This modulation may correspond, depending on the case and in particular on the nature of the biological marker, to an increase or a decrease in the expression of said marker. In particular, the expression of inflammatory markers is known to be increased when the skin is damaged, for example by exogenous stress.In contrast, the expression of these inflammation markers is not affected by well-tolerated active ingredients or formulations. In a preferred embodiment, the biological marker in step b) is an inflammation marker, for example, one of those described here.

[0129] Preferably, tolerance consists of maintaining the equilibrium of the microbial component. By "maintaining equilibrium" is meant that at least one microorganism, preferably at least one genus or species of bacteria, is maintained in the microbial component of the model following application of the cosmetic agent or formulation in step a). Preferably, the abundance of said at least one microorganism is not significantly altered following application of the cosmetic agent or formulation in step a). In order to determine whether said at least one microorganism is maintained, or at a level that is not significantly different, the presence of the microorganism, or its level of abundance, is compared to a reference condition or level, for example in a model in which the agent has not been applied or in normal skin in vivo.

[0130] The invention also relates to a kit comprising a microbial component and a lipid component intended to be applied to a skin substitute, the microbial component comprising at least four genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium.

[0131] Preferably, the microbial component comprises at least five, six, seven, eight, nine, or ten genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium, more preferably all the genera described above. Preferably, the at least four genera comprise the following genera: Cutibacterium, Staphylococcus, Streptococcus, and Burkholderia. The microbial component may also comprise one or more genera or species of fungus and / or virus.

[0132] Preferably, the microbial component corresponds to the skin microbiota of at least one subject. "Microbiota" refers to the collection of microorganisms present in an environment. The skin microbiota therefore corresponds to the collection of microorganisms present on the skin. Of course, the composition of the microbiota can vary depending on the skin site. Preferably, the cutaneous microbial component corresponds to the skin microbiota of the face, preferably the forehead, of at least one subject. Also, according to a preferred embodiment, the cutaneous microbial component is obtained from a sample of at least one subject, more preferably from the face and even more preferably from the forehead. Preferably, the microbial component has a viability of 5,000 RLU.

[0133] The lipid component is as described above.

[0134] Preferably, the kit also includes an instruction manual.

[0135] Preferably, the kit further comprises at least one chemical component such as described here. Preferably, the kit includes an allergen, a pollutant, a surfactant, a solvent, or at least a pro-inflammatory stimulating reagent such as a cytokine, or cortisol.

[0136] Said kit advantageously allows obtaining the model of reconstituted skin. EXAMPLES Example 1: Sebum and microbiota preparation

[0137] - Sebum preparation: Sebum was collected from the foreheads of 10 volunteers with no apparent skin pathology using a standard collection kit. Each collection tube was then washed three times with hexane to extract the sebum. The extracts were then combined, and the solvent was evaporated under vacuum. The mass of the collected sebum was checked (e.g., 1100 mg for one preparation), and then the sebum was resuspended in 20 mL of hexane. One hundred vials of sebum were prepared, with 9 mg of substance per vial, and packaged under argon. For deposition of the sebum onto the reconstructed epidermis, the vials were then resuspended with 250 µL of absolute ethanol (Fischer Scientific, Illkirch, France) and then diluted 1 / 20th in physiological saline. The sebum was characterized by GC / MS ([Fig. 1]). - Microbiota preparation: The microbiota was collected from 18 volunteers without apparent skin pathology using a swab soaked in physiological saline (Fischer Scientific, Illkirch, France) containing 0.1% by volume of Triton X-100 (Fischer Scientific, Illkirch, France). The swab was vigorously rubbed on the donor's forehead for 4 seconds. Each swab was then placed in a tube for centrifugation. After centrifugation Following the fumaration, a colorless liquid (approximately 60 qL) is collected and its volume is adjusted to 120 qL with physiological saline. Each microbiota is then mixed with sebum to facilitate deposition. 22 qL of the sebum / microbiota mixture is deposited onto the surface of a reconstructed human epidermis and spread using a finger cot. A given sebum / microbiota mixture can be applied to up to 6 reconstructed epidermis. The reconstructed human epidermis is incubated at 32°C, 5% CO2, and 60% humidity.

[0138] The incubation time is set at 48 h, which allows for a sufficient quantity of bacteria without damaging the reconstructed human epidermis.

[0139] On the face, quantities of sebum are found ranging from >66qg / cm2 for oily skin, 33-66qg / cm2 for normal skin and <33qg / cm2 for dry skin.

[0140] The inventors deposited 24qg of sebum on reconstructed epidermis of 0.6cm2, which corresponds to a concentration of 40qg / cm2.

[0141] Regarding the microbiota, according to the literature, there are approximately 1 million bacteria with hundreds of different species found per square centimeter. These quantities vary depending on the area studied. At the end of 48 hours, approximately 100,000 RLUs are found per 0.6 cm² RHE, which corresponds to a growth of 1 to 2 log.

[0142] Example 2: Establishment of the reconstituted skin model with or without the addition of stress

[0143] Methods

[0144] The epidermis was reconstructed from skin excisions obtained from cosmetic surgery according to the method described by Frankart et al. (Frankart et al., Exp. Dermatol. 2012,21(11), 871-875).

[0145] Briefly, the cells (keratinocytes) were isolated from the skin excisions, then cultured before being seeded onto culture inserts immersed in culture medium, then the culture inserts are placed at the air / liquid interface in an incubator at 37°C in a humidified atmosphere with 5% CO2, to form the stratum corneum.

[0146] At 14 days (D14), a reconstructed epidermis with a surface area of ​​0.6 cm² is formed. The culture medium is renewed every 24 hours.

[0147] On day 10, the reconstructed epidermis is placed in a culture medium without anti- On day 13, the reconstructed epidermis is placed in 12-well plates with antibiotic-free maintenance medium (see Frankart et al., 2012) and phenol red. After 2 hours, a sebum / microbiota mixture, obtained as described in Example 1, is deposited on the surface of each reconstructed human epidermis and spread using a finger cot. The reconstructed human epidermis is then inoculated with a microbiota inoculum with a viability of approximately 5000 RLU.

[0148] Epidermal survival is performed under sterile conditions. Each insert containing an epidermis is placed in a well of a new 12-well plate containing the new maintenance medium alone or supplemented to induce stress. The plate is incubated at 32°C under 5% CO2 and 60% relative humidity. The culture medium is changed every 24 hours up to 48 hours of incubation.

[0149] In order to mimic an inflammatory stress of the atopic dermatitis type, the medium used is the maintenance medium (1 mL / well) supplemented with interleukin 1b at 0.06pg / mL, the TLR ligand (Poly (1:C) at 10g / ml, and Pam3CSK4 at 5pg / ml). Microbiota viability measurement

[0150] Upon receipt, microbiota viability is measured using the Promega BacTiter ATP Lite kit, following the supplier's protocol. Microbiota viability after culture is evaluated using the same kit. The viability measurement is a bioluminescent assay involving luciferase. The amount of light emitted is proportional to the amount of ATP present in the sample and therefore to the number of live cells. To monitor bacterial growth during the experiment, a viability reading is performed in duplicate on the microbiota collected from each epidermis (Promega BacTiter-Glo Microbial Viability Cell Assay kit). The ATP mix and the microbiota are placed in each well in a 1:1 ratio. After application, the plate (Greiner Low Binding 655209) is shaken for 30 seconds at 500 rpm and then incubated at room temperature for 5 minutes. The reading is then performed using a Clariostar plate reader. DNA extraction from the microbiota

[0151] The DNA of microorganisms before and / or after growth on reconstructed epidermis is extracted using a Qiacube following the QIAamp DNA Investigator Kit protocol from Qiagen.

[0152] Absolute quantification of microorganisms of interest by ddPCR

[0153] Droplet Digital PCR (ddPCR) is a digital PCR technique based on the generation of droplets using a water / oil emulsion. From a sample, it generates more than 20,000 droplets within which PCR amplification is performed. After amplification, each droplet is analyzed to quantify the number of positive droplets in the sample. This technique has the particular advantage of allowing the amplification of DNA present in small quantities in a sample.

[0154] ddPCR is performed in a 96-well plate: 5 pL of sample are added to 15 pL of ddPCR mix as explained in the Bio-Rad protocol. The probes used correspond to the species being sought, e.g., S. epidermidis, S. aureus, C. acnes.

[0155] The generation of droplets is carried out using the QX200 Droplets Generator device The plate is then placed in the thermocycler for amplification using Biorad's QX200 Droplet Reader and analyzed with QuantaSoft software. Targeted Sequencing

[0156] The impact of "culture on reconstructed human epidermis" on the microbiota is assessed by targeted sequencing.

[0157] Sequencing was performed on a MiSeq sequencer (Illumina) with primers adapted to each target. Sequence preprocessing was carried out using a computer pipeline developed by INRAE ​​running under Mothur (version 1.44.0). 16S Bacterial Sequencing

[0158] The primers used target the variable regions V1-V3 of the 16S ribosomal RNA sequence of prokaryotes (bacteria and archaea). Sequence preprocessing is performed by a computer pipeline developed by INRAE ​​running under Mothur (version 1.44.0).

[0159] Barcodes, primers, and chimeras were removed from the sequence files during the filtering steps. Sequences exhibiting 100% homology were grouped into unique sequences, then into OTUs (operational taxonomy units: 97% threshold) which will be identified subsequently.

[0160] Bioinformatic analysis of sequencing data allows the identification of microorganisms present at different taxonomic levels (phylum, genus (e.g., for Staphylococcus), and species (e.g., for C. acnes)). Phylogenetic affiliation analysis down to the genus level was performed using bioinformatics tools for processing large amounts of sequence data (Mothur). Identification was based on the Greengenes taxonomy for bacteria.

[0161] Sequencing of target Staphylococcus species “Tuf”

[0162] The primers used target the TUF gene. Bioinformatic analysis of the sequencing data allows for the identification of Staphylococcus species present at different taxonomic levels (genus and species are targeted). Phylogenetic affiliation analysis down to the species level was performed using bioinformatics tools for processing large amounts of sequence data (Mothur). Identification was performed against the Staphylococcus list.

[0163] Evaluation of the impact of cultured microbiota on healthy and / or stressed reconstructed epidermis

[0164] Expression of genes encoding barrier function proteins (Claudin 1, Filaggrin and TGM1) or human antimicrobial peptides (DEFB4A and S100A7) has was assessed by RT-qPCR. DNA extraction

[0165] The reconstructed epidermal halves are stored in 300 pL of a 1% [3-mercaptoethanol] RLT solution (Qiagen) and frozen at -80°C pending extraction. Initially, the RHEs are separated from their membrane using forceps. To mechanically lyse the cells, a 5 mm Stainless Steel Bead (Qiagen) is then added to each tube, and the tubes are then run through the Tissue Lyser (Qiagen) for 2 minutes (2x) at 20 Hz. Subsequently, the RNA is extracted according to the Qiagen RNA Fibrous Tissue Mini Kit extraction protocol. Extractions are performed using Qiacube (Qiagen). The amount of RNA extracted is measured using Nanodrop. RT-qPCR

[0166] The RNA volume is then adjusted to reverse-transcribe 1 pg of RNA per sample. 4 pL of VILO MasterMix SuperScript are then added to the samples.

[0167] The cDNA obtained are then diluted by a factor of twentieth and arranged in a 384-well plate (triplicates) using the Qiagen EpMotion robot. 5.5 pL of reaction mix (5 pL of TaqMan Fast Advanced Master Mix + 0.5 pL of probes per reaction) are then added to each well.

[0168] GAPDH, YWHAZ and UBC are the reference genes chosen for qPCR analysis. IL-8 and TSLP assay by ELISA

[0169] LTL-8 is measured in culture supernatants (24H, 48H, and 72H) using the Bioplex IL-8 ELISA kit from R&D Bio-Techne. The supernatants are measured in triplicate. Plate reading (Costar 3590 Corning) is performed on the Clariostar plate reader. Similarly, TSLP is measured in the supernatants using the Bioplex TSLP kit from R&D Bio-Techne. Results

[0170] Growth of the microbiota in the reconstructed skin model

[0171] Monitoring viability during the implementation time of this model reports of the growth of the microbiota on the epidermis.

[0172] The results of microbiota growth on healthy or inflamed epidermis are presented in Table 2 below.

[0173] Microbiota growth on epidermis assessed by the difference between viability at 48h and viability at T0. NS: not significant; S: significant

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] [Tables 2] Viability (T48h-T0) P-value Statistical significance Healthy epidermis 72,814 0.8252 NS Inflammatory epidermis 1,003,647 0.0036 S Microbiota growth on complete epidermis is 1 to 2 log in 48 hours. Significantly greater growth is observed on inflamed epidermis than on healthy epidermis. While not theoretically supported, it is possible that the barrier is compromised in the inflamed epidermis, allowing microorganisms access to additional nutrients and thus enabling greater growth. Microbiota component analysis The bacterial composition of the microbiota was assessed at day 13 (before inoculation on the model). At day 13, the bacterial genera identified in the microbiota are as follows: Cutibacterium (more particularly C. acnes), Staphylococcus, Streptococcus, Bur-kholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylo-bacterium and Brevibacterium. The Staphylococcus species have been more precisely identified. The Staphylococcus species identified in the microbiota are: S. epidermidis, S. capitis, S. wameri, S. hominis and S. aureus. Among the bacterial genera identified in the microbiota at 48 hours, Cutibacterium and Staphylococcus were notably detected. The Staphylococcus species were more precisely identified. The Staphylococcus species identified in the microbiota at 48 hours are the same as those initially present (S. epidermidis, S. capitis, S. aureus, S. wameri, S. hominis). Evaluation of the impact of microbiota / sebum on healthy epidermis. Effect of microbiota / sebum on the secretion of inflammatory cytokines by healthy epidermis. 1 / IL-8 Secretion The effects of microbiota / sebum on IL-8 secretion are summarized in Table 3. IL-8 production kinetics as a function of microbiota / sebum growth time on epidermis.

[0185] [Tables3] Healthy epidermis Mean (pg) of IL-8 secreted per culture well, n=18 Time (H) Without microbiota / sebum With microbiota / sebum 24 46.32 338.18 48 69.9 620.78

[0186] The presence of microbiota / sebum on healthy epidermis leads to the production of IL-8; therefore, there is a reaction of the epidermis to the presence of microorganisms. 2 / Secretion of TSLP

[0187] TSLP is an inflammatory cytokine characteristic of atopic dermatitis. Its secretion is markedly stimulated in atopic dermatitis and promotes the initiation of an inflammatory response by Th2 cells. It is measured in the supernatant after 24 hours of culture.

[0188] Healthy epidermis produces little TSLP.

[0189] The effects of microbiota / sebum on TSLP secretion are summarized in Table 4.

[0190] Production of TSLP by healthy epidermis with and without microbiota / sebum

[0191] [Tables4] Healthy epidermis Mean (pg) of TSLP secreted per mL of culture Time (H) Without microbiota / sebum (n=3) With microbiota / sebum (n=18) 24 3.08 3.6

[0192] TSLP production is not modified by the presence of microbiota on healthy epidermis.

[0193] 3 / Effects on the expression of antimicrobial peptides

[0194] The DEFB4A gene encodes [3-defensin 2], a skin antimicrobial peptide secreted by keratinocytes. The production of antimicrobial peptides is stimulated by the presence of microorganisms. S100A7 encodes the protein known as psoriasin, which has antimicrobial properties.

[0195] The effects of microbiota / sebum on the expression of two antimicrobial peptides are presented in Table 5.

[0196] Expression of DEFB4A and S100A7 by healthy epidermis with and without microbiota

[0197] [Tables5] Healthy Epidermis Time (H) DEFB4A gene expression compared to healthy epidermis without microbiota / sebum (RQ) Without microbiota / sebum (n=3) With microbiota / sebum (n=18) 48H 1 3.03 (p=0.14) S100A7 gene expression compared to healthy epidermis without microbiota / sebum (RQ) Without microbiota / sebum (n=3) With microbiota / sebum (n=18) 48H 1 4.75

[0198] RQ: "Relative quantification", RQ > 2: overexpression, 0.5 < RQ: underexpression

[0199] The presence of microbiota / sebum on healthy epidermis leads to a slight increase The expression of the antimicrobial peptides DEFB4A and S100A7 by the epidermis is affected. The presence of microbiota / sebum on healthy epidermis stimulates an "innate" immune response.

[0200] 4 / Effects on the expression of barrier function genes

[0201] In order to determine whether the microbiota / sebum has an effect on the barrier function of healthy epidermis, the inventors quantified by RT-qPCR the expression of the gene encoding Claudin-1, a protein involved in tight junctions, and the expression of the gene encoding Filaggrin.

[0202] The TGM1 gene codes for transglutaminase-1, an enzyme involved in the formation of the stratum comeum and barrier function.

[0203] The effects of microbiota / sebum on TGM1 expression are shown in Table 6.

[0204] Expression of TGM1 by healthy epidermis with and without microbiota

[0205] [Tableauxô] Healthy epidermis Time (H) TGM1 gene expression compared to healthy epidermis without microbiota / sebum (RQ) Without microbiota / sebum (n=3) With microbiota / sebum (n=18) 48H 1 2.1 (p=0.028)

[0206] RQ > 2: overexpression, 0.5 < RQ: underexpression

[0207] The presence of microbiota / sebum on healthy epidermis does not lead to a significant change in the expression of genes encoding the proteins involved in the barrier function, Filaggrine and Claudine 1 (results not shown). The expression of the gene encoding the TGM1 enzyme is increased.

[0208] - Evaluation of the impact of the microbiota on inflamed epidermis 1 / IL-8 secretion

[0209] The effects of the microbiota on IL-8 secretion are summarized in Table 7.

[0210] Illustrative example of IL-8 production at 24h and 48h on inflamed epidermis

[0211] [Tables?] Inflammatory epidermis Mean (pg) of IL-8 secreted per culture well, n=18 Time (H) Without microbiota / sebum With microbiota / sebum 24 2020.04 3712.16 48 4091.36 6884.42

[0212] IL-8 production is multiplied by 1.7 to 2.2 times. The presence of microbiota / sebum on inflamed epidermis leads to a significant increase in IL-8 production, which accounts for an amplification of the inflammatory reaction of the epidermis in the presence of microbiota.

[0213] Similar results are described in the literature in patients with atopic dermatitis (Casas et al., 2008, Skin Pharmacology and Physiology 21, 260-268). Furthermore, IL-8 production is increased 2- to 10-fold in the inflammatory model compared to the "healthy" model (without added inflammatory stress). These results are also similar to those described in Casas et al., 2008, where IL-8 production was increased by a factor of 4.3 (2087 / 487, see Table 3).

[0214] These results illustrate that the model according to the invention, comprising a microbiota and sebum, is representative of what happens in vivo, particularly under inflammatory conditions. 2 / TSLP Secretion

[0215] The effects of microbiota / sebum on TSLP secretion are summarized in Table 8.

[0216] Production of TSLP by inflamed epidermis with and without microbiota

[0217] [Tables8] Inflammatory epidermis Mean (pg) of TSLP secreted per mL of culture (n=18) Time (H) Without microbiota / sebum With microbiota / sebum 24 49.28 34.63

[0218] The production of TSLP by inflamed epidermis allows us to validate this model.

[0219] Indeed, as expected, TSLP is produced under inflammatory conditions compared to the "healthy" model (without added inflammatory stress), but the presence of microbiota / sebum does not significantly alter TSLP production.

[0220] 3 / Effects on the expression of antimicrobial peptides

[0221] The effects of the microbiota on the expression of two antimicrobial peptides are presented in Table 9.

[0222] Expression of DEFB4A and S100A7 by inflamed epidermis with and without microbiota / sebum.

[0223] [Tables9] Inflammatory Epidermis Time (H) DEFB4A gene expression compared to healthy epidermis without microbiota / sebum (RQ) Without microbiota / sebum (n=3) With microbiota / sebum (n=18) 48H 16.63 (p<0.023) 117.05 (p=0.009) S100A7 gene expression compared to healthy epidermis without microbiota / sebum (RQ) Without microbiota / sebum (n=3) With microbiota / sebum (n=18) 48H 62.7 (p<0.00001) 86.03 (p=0.001)

[0224] Expression relative to the control healthy epidermis without microbiota. RQ > 2: overexpression, 0.5 < RQ: underexpression

[0225] The presence of microbiota / sebum on inflamed epidermis leads to a significant increase in the expression of the antimicrobial peptides DEFB4A and S100A7 by the epidermis. As expected, an exacerbated sensitivity of the inflamed epidermis is observed in the presence of microbiota / sebum.

[0226] 4 / Effects on the expression of barrier function genes

[0227] The effects of microbiota / sebum on TGM1 expression are presented in Table 10.

[0228] Expression of TGM1 by inflamed epidermis with and without microbiota / sebum.

[0229] [TableauxlO] Inflammatory epidermis Time (H) TGM1 gene expression compared to healthy epidermis without microbiota / sebum (RQ), (n=18) Without microbiota / sebum With microbiota / sebum 48H 1.8 (p=0.151) 5.97 (p=0.003)

[0230] Calculation of RQs relative to gene expression in healthy epidermis without microbiota. RQ > 2: overexpression, 0.5 < RQ: underexpression

[0231] The presence of microbiota on inflamed epidermis leads to a significant increase in the expression of TGM1 by the epidermis, which confirms an exacerbated sensitivity of the inflamed epidermis in the presence of microbiota.

[0232] In light of the above results, the inventors clearly demonstrate the value of the reconstituted skin model comprising a skin substitute as well as a microbial component and a lipid component. Indeed, under non-pathological conditions, the presence of a microbial component and a lipid component induces the production of certain inflammatory markers such as IL-8, and a slight increase in the expression of antimicrobial peptides, thus stimulating innate immunity. This suggests that the microbiota induces a baseline level of inflammation under these conditions. Furthermore, under conditions of chemical and inflammatory stress, the microbiota induces an exacerbated inflammatory response, leading to impaired barrier function. Thus, in the presence of inflammatory chemical stress, the model advantageously reproduces atopic dermatitis-type inflammation.

[0233] This model is therefore perfectly suited to evaluate new assets that would be potentially useful in the prevention or treatment of atopic dermatitis.

[0234] This model is also suitable for evaluating new assets that would be potentially useful in the prevention or treatment of inflammatory dermatoses, such as psoriasis, acne, seborrheic dermatitis, chronic urticaria, eczema, rosacea.

[0235] Finally, this integrated model is particularly interesting for testing new assets with a putative activity in the protection, or even the strengthening, of the barrier function.

[0236] Example 3: Evaluation of the effect of a stress simulating sun exposure on the reconstructed skin model and the effect of a sunscreen

[0237] Physical stress is applied to the reconstructed skin model comprising microbiota and sebum. Specifically, the model is exposed to UV radiation, thus mimicking sun exposure. A sunscreen, 5,6,5',6'-tetraphenyl-3,3'-(l,4-phenylene)-bis[l,2,4]triazine, formulated (Formulation A, described in Table 10 below), is applied 1 hour before the radiation exposure to study its protective effect on this integrated model.

[0238] Table 11: Formulation A Formulation A

[0239] [Tables] Ingredients % (w / w) 5,6,5',6'-tetraphenyl-3,3'-(l,4-phenylene)-bis[l,2,4]triazine 3-3.5 Glycerin 5.0 Demineralized water qs 100 Xanthan gum 0.2 C12-C15 alkyl benzoate 12.0 Dicaprylyl carbonate 9.0 Caprylic / capric triglycerides 9.0 Preservatives qs Stearyl alcohol 1.0 Glyceryl monostearate 0.5 Glyceryl behenate 0.4 PEG-100 stearate 0.5 VP / eicosene copolymer 1.0 Polyacrylate 0.2 Potassium cetyl phosphate 2.0 Methods

[0240] The model described in Example 2 was used.

[0241] Eighteen reconstructed epidermis (corresponding to 3 donors with six reconstructed epidermis obtained per donor) with microbiota from 18 distinct donors and sebum are used per condition (untreated / unirradiated, untreated / irradiated, Formulation A / irradiated, Formulation A / unirradiated). Exogenous stress is performed 24 hours after the addition of the microbiota / sebum, i.e., on day 14.

[0242] The reconstructed epidermis is irradiated by simulated solar radiation in using a Suntest CPS+ ATLAS chamber (Material Testing Technology BV, Moussy le Neuf, France) equipped with an NXE 1500 xenon lamp and fitted with a UV filter to eliminate wavelengths below 290 nm. Irradiance in the UV spectrum is approximately 70 W / m² from 290 to 400 nm. Skin models are exposed to a single acute UV dose of 16.5 J / cm² (approximately 45 min), and the irradiation chamber is maintained at 37 °C using ice water and an airflow.

[0243] Formulation A is applied at a dose of 2 mg / cm² to the surface of the reconstituted skin model 2 hours before exposure to radiation. After irradiation with the full solar spectrum (up to 790 nm), the model is maintained in culture for 24 hours at 32 °C and 60% humidity.

[0244] At the end of the experiment, the surface of the reconstructed epidermis is washed twice with 100 ml of physiological saline, and then dried with a cotton swab. This step aims to recover the skin microbiota as well as lipids (here sebum) for analysis separately from the skin cells. Metabolomics approach

[0245] At the end of the study, the reconstructed epidermis was separated from the insert and weighed to standardize the results obtained with the metabolomics approach. The reconstructed epidermis was ground, and 108 samples were extracted. Briefly, the samples were ground in 2 x µL of acetonitrile / water 1 / 9 (v:v) using a fastprep with Lysing Matrix M tubes, 6 cycles of 20 seconds at strength 4, with 2 min on ice between each cycle. A volume of 800 pL of each sample was evaporated to dryness with a SpeedVac and then normalized in 220 pL of D₂O. The samples were analyzed by NMR and then mass spectrometry.

[0246] The 1H NMR spectra are obtained at 300 K on a Bruker Avance III HD 600 MHz NMR spectrometer (Bruker Biospin, Rheinstetten, Germany), operating at 600.13 MHz for the 1H resonance frequency, using a 5 mm 1H-13C-15N-31P inverse-detection cryosprobe attached to a Cryoplatform (the pre-amplification unit). The 1H NMR spectra are acquired using the NOESY 1D experiment with pre-saturation for water suppression (noesyprld), with a mixing time of 100 ms.

[0247] After NMR analysis, the samples are transferred from the NMR tubes to the UHPLC bottles. The samples are centrifuged at 9000 g for 5 min. A volume of 10 pL is then injected into the Waters ACQUITY UHPLC system (Manchester, UK), using water / methanol / acetic acid 95 / 5 / 0.1 (v:v:v) as mobile phase A and methanol / acetic acid 100 / 0.1 (v:v) as mobile phase B, at a flow rate of 0.3 mL / min. The following gradient is used: 0–30 min: 0% to 100% of B, 30–34 min: 100% of B. Separation is carried out at 30°C with a Thermo Scientific Hypersil Gold C18 column (100 x 2.1 mm, 1.9 pm) (Les Ulis, France). The following electrospray parameters are applied: capillary voltage of 0.5 kV, sampling cone voltage of 30 V, source temperature of 120°C, desolvation temperature of 350°C, cone gas flow rate of 50 L / h, and desolvation gas flow rate of 600 L / h in positive mode; capillary voltage of 0.5 kV, sampling cone voltage of 30 V, source temperature of 120°C, desolvation temperature of 550°C, cone gas flow rate of 30 L / h, and desolvation gas flow rate of 600 L / h in negative mode. High-resolution mass spectra are acquired with a Waters Synapt G2-Si mass spectrometer (Manchester, UK), between m / z 50 and 800 in sensitivity and centroid modes. The samples are analyzed randomly, and a QC sample corresponding to a pool of all the samples is analyzed 11 times along the sequence.

[0248] Structural identifications of the discriminating metabolites are performed on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Les Ulis, France) coupled to a U3000 liquid chromatography system (Thermo Scientific, Les Ulis, France). Data analysis

[0249] Statistical analysis and network mapping are carried out by the MetaboHUB-MetaToul-AXIOM platform.

[0250] The data are normalized to allow the quantification of signals detected in several samples.

[0251] Principal component analysis is first applied to verify the validity of the acquisition, to detect potential outliers and internal clusters.

[0252] In a biological context, confounding factors are commonly encountered: these factors add noise to the data, corresponding to unwanted variability. Several sources of noise are possible: experimental, instrumental, etc. When the variability due to these factors is greater than the variability due to the factor of interest, no discrimination between treatment groups can be found. Discriminant analysis using orthogonal partial least squares (O-PLS-DA) aims to eliminate this unwanted variation. A permutation test is performed to evaluate the robustness of the PLS-DA models. The value of the Importance on Projection (VIP) variable (threshold = 1) was used to find discriminant features.

[0253] For each discriminating characteristic, a univariate Wilcoxon test and a false discovery rate (FDR) correction are performed to take into account multiple tests (FDR-corrected p-value threshold = 0.05).

[0254] Multivariate analyses were performed using SIMCA vl5 software (Umetrics, Umeå, Sweden). The mixOmics package (Rohart et al., 2017) was used to perform multilevel analyses.

[0255] To create the metabolic network, freely accessible software is developed and Used by INRAE, Toxalim: MetExplore. This is a web server that allows the linking of metabolites identified in untargeted metabolomics experiments within the context of genome-wide reconstructed metabolic networks. The analysis pipeline includes mapping metabolomics data onto an organism's specific metabolic network, followed by the application of graph-based methods and advanced visualization tools to enhance data analysis. Results :

[0256] Pharmacological analysis of pathways and metabolites 1 / Oxidative stress: a metabolomics approach

[0257] The following table reports the observed variations (Log 2 of variations) under the effect of UV irradiation for different discriminating metabolites of oxidative stress in the epidermis of the reconstituted skin model.

[0258] [Tables 12] MS Negative NI CTRL vs. IR CTRL NI FA vs. IR FA Uric acid 2.62 1.34 Gluthation -9.03 -1.77 MS Positive NI CTRL vs. IR CTRL NI FA vs. IR FA Xanthine 0.57 ns Glutathione -6.83 -1.61

[0259] NI: Non-irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; ns: Not significant

[0260] A significant modulation of these metabolites is observed after exposure to radiation, with a marked increase in xanthine and uric acid and a significant decrease in glutathione. These results are consistent with in vivo studies on human volunteers (Randhawa et al., PLoS One. 2014; 9(3): e90367). These results demonstrate the validation of the model according to the invention for studying sun exposure and predicting the in vivo effect on human skin.

[0261] Furthermore, these results demonstrate a significant protective effect of a formulation containing phenylene bis-diphenyltriazine (Formulation A) against oxidative stress due to sun exposure. The model also allows for the identification or evaluation of the in vitro efficacy of an active ingredient to prevent and / or treat at least one skin disorder induced by at least one exogenous stressor such as UV irradiation.

[0262] 2 / Barrier function and hydration: a metabolomics approach

[0263] The following table reports the observed variations (Log 2 of variations) under the effect of UV irradiation for different discriminating metabolites of the barrier function and hydration of the skin, in the epidermis of the model.

[0264] [Tables 13] NMR NI CTRL vs. IR CTRL NI FA vs. IR FA Octanoic acid 1.77 0.72 Serine 0.34 Ns Glutamate -0.52 -0.18 Phosphory Icholine -0.41 -0.27 MS Negative NI CTRL vs. IR CTRL NI FA vs. IR FA Histidine -1.82 -0.77 L-Glutamine -2.35 -0.85 MS Positive NI CTRL vs. IR CTRL NI FA vs. IR FA L-Alanine -2.79 -1.44 Methionine -1.02 -0.48 Xanthine 0.57 Ns

[0265] NI: Non-irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; Ns: Not significant

[0266] These results demonstrate that topical application of a formulation including phenylene bis-diphenyltriazine (Formulation A) makes it possible to very significantly dampen the imbalances induced by UV radiation, and thus to preserve the barrier function and hydration of the skin. 3 / Microbiota: a metabolomics approach

[0267] The following table reports the observed variations (Log 2 of variations) under the effect of UV irradiation for different metabolites detected on the surface of the model in relation to the microbiota of the model.

[0268] [Tables 14] NMR NI CTRL vs. IR CTRL NI FA vs. IR FA Formate 0.68 ns Acetate 0.69 ns Glucose + maltose 1.60 ns Glycerol 1.55 ns Betaine 0.64 ns Octanoate 1.06 ns MS Positive NI CTRL vs. IR CTRL NI FA vs. IR FA Tyrosine 1.70 ns Panthenol 0.71 ns Phenylalanine 1.52 ns

[0269] NI: Non-irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; ns: Not significant

[0270] UV irradiation induces significant changes in a large number of metabolites.

[0271] These results also demonstrate that topical application of a formulation containing phenylene bis-diphenyltriazine (Formulation A) significantly reduces imbalances induced by UV radiation, thereby maintaining the balance of the skin's microbial component. Phenylene bis-diphenyltriazine thus exhibits a true symbiotic action. 4 / C. acne

[0272] C. acnes is a very abundant bacterial species in the model microbiota.

[0273] The inventors have demonstrated a significantly increased abundance under the effect of irradiation (see Table 15).

[0274] [Tables 15] IR CTRL vs. NI CTRL IR FA vs. NI FA C. acnes ++ (3.57% / 1.59%) p=0.0069 ns

[0275] NI: Non-irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; ns: Not significant

[0276] These results demonstrate that topical application of a formulation comprising phenylene bis-diphenyltriazine (Formulation A) significantly reduces imbalances induced by UV radiation, and thus protects the mi- crobiote and to maintain the balance of the skin's microbial component.

[0277] Thanks to this model of human epidermis reconstructed with microbiota and sebum, the inventors identified a very precise metabolomic signature of the skin exposed to UV radiation, mimicking sun exposure.

[0278] This integrated model is particularly interesting because it allows us to get closer to the conditions observed in vivo.

[0279] In addition, the inventors have shown not only that the effects of different stresses can be observed at the metabolomic level but also that Formulation A makes it possible to protect the skin ecosystem from sun exposure and to preserve the symbiosis and homeostasis of the skin.

[0280] These results confirm the validity of the reconstructed skin model. These results also confirm the validity of the reconstructed skin model for use in screening methods or for evaluating the efficacy of cosmetic active ingredients in preventing or treating skin disorders, as well as in methods for evaluating the tolerability of an active ingredient or cosmetic formulation. Most advantageously, the effect of exogenous stress or an active ingredient could be determined based not only on a single marker but on several markers, making it possible to establish a metabolomic signature and demonstrating the pathophysiological relevance of the reconstructed skin model.

Claims

Demands

1. A reconstituted skin model comprising: - a skin substitute, and - a microbial component and a lipid component on the surface of said skin substitute. Said microbial component comprising at least four genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium, and Brevibacterium, preferably at least the genera Cutibacterium, Staphylococcus, Streptococcus, and Burkholderia.

2. Model according to claim 1, characterized in that the lipid component comprises a mixture of free fatty acids, triglycerides, squalene, wax and / or wax esters, and cholesterol and / or cholesterol esters.

3. Model according to claim 2, characterized in that the lipid component comprises 30 to 50% w / w of triglycerides, 15 to 30% w / w of free fatty acids, 25 to 30% w / w of waxes and / or wax esters, 12 to 20% w / w of squalene and 4.5 to 8.5% w / w of cholesterol and / or cholesterol esters.

4. Model according to any one of claims 1 to 3, characterized in that the lipid component is taken from a sample of at least one subject, preferably from the face.

5. Model according to any one of claims 1 to 4, characterized in that the microbial component is a cutaneous microbial component taken from a sample of at least one subject, preferably from the face and even more preferably from the forehead.

6. A method for obtaining a reconstituted skin model according to any one of claims 1 to 5, comprising a) Providing a skin substitute, b) Applying to said skin substitute, concomitantly or sequentially, a microbial component and a lipid component.

7. A method according to claim 6 characterized in that the reconstituted skin is stressed and in that the method further comprises a step c) Performing at least one exogenous stress.

8. A method according to claim 7 characterized in that the exogenous stress is a chemical or physical stress.

9. The method according to claim 8 characterized in that the physical stress is irradiation in the UV range including UVA and / or UVB; and / or visible light including high energy blue visible light and / or infrared.

10. A method according to claim 8 characterized in that chemical stress is a pro-inflammatory stimulation reagent or cortisol.

11. A screening method for the in vitro identification of an active ingredient or cosmetic formulation to prevent and / or improve at least one skin disorder induced following at least one exogenous stress, comprising the following steps: a) Applying said active ingredient or cosmetic formulation to the reconstituted skin model according to any one of claims 1 to 5; b) Performing at least one exogenous stress on said model; c) Measuring at least one expression level of at least one biological marker; and d) Determining whether said active ingredient or cosmetic formulation prevents and / or improves at least one skin disorder based on at least one expression level measured in step c).

12. Method for evaluating the in vitro efficacy of an active ingredient or cosmetic formulation for preventing and / or treating at least one skin disorder induced following at least one exogenous stress, comprising the following steps: a) Applying said active ingredient or cosmetic formulation to the reconstituted skin model according to any one of claims 1 to 5; b) Performing at least one exogenous stress on said model; c) Measuring at least one level of expression of at least one biological marker; and d) Evaluating the efficacy of said active ingredient or cosmetic formulation as a function of at least one level of expression measured in step c).

13. A method according to claim 11 or 12, wherein at least one skin disorder is selected from disorders related to aging, pollution, radiation; inflammatory dermatoses, preferably atopic dermatitis, acne, psoriasis; rosacea; pruritus; impaired barrier function; reactive skin; xerosis.

14. Method according to claim 12 of evaluating the in vitro efficacy of at least one sunscreen or cosmetic formulation to prevent and / or treat at least one skin disorder induced following at least one exogenous stress consisting of UV, infrared, or blue light irradiation.

15. Method for evaluating in vitro the tolerance of an active ingredient or cosmetic formulation, comprising the following steps: a) The application of said active ingredient or cosmetic formulation to the reconstituted skin model according to any one of claims 1 to 5; b) At least one measurement of the level of expression of at least one biological marker in the skin model of step a); and c) Evaluating whether said active ingredient or cosmetic formulation is well tolerated by the skin model.

16. Method according to claim 15, characterized in that the tolerance consists of maintaining the balance of the microbial component.

17. Kit comprising a microbial component and a lipid component intended to be applied to a skin substitute, the microbial component comprising at least four genera of microorganisms selected from the genera Cutibacterium, Staphylococcus, Strep-tococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Cory-nebacterium, Methylobacterium, and Brevibacterium.

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