Reconstituted-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, and also to a method for obtaining the reconstituted-skin model. The reconstituted-skin model can in particular be used in a screening method for the identification in vitro of an active agent or of a cosmetic formulation for preventing and / or improving at least one skin disorder induced following at least one exogenous stress; a method for the evaluation of the efficacy in vitro of an active agent or of a cosmetic formulation for preventing and / or treating at least one skin disorder induced following at least one exogenous stress; or an evaluation in vitro of the tolerance with respect to an active agent or a cosmetic formulation.

Description

[0001] RECONSTRUCTED SKIN MODEL

[0002] Field of invention

[0003] The field of the invention relates to a new skin model integrating 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, to this integrated model, physical or chemical stress can be added to reproduce lesions or pathological situations, which can cause imbalances in the skin microbiota.

[0004] State of the art

[0005] The skin is much more than an external envelope; it is in fact a true organ necessary for life. It constitutes a platform for exchange between our internal environment and the external environment and is therefore exposed to multiple attacks and variations to which it constantly adapts. The skin therefore ensures, among other things, a true barrier function, previously 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 divided into 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, builds up an energy reserve, and thermoregulates the body. The dermis is the layer between the hypodermis and the epidermis. It is the thickest layer of the skin.The dermis houses blood and lymphatic vessels, nerves, sweat glands that produce sweat, and pilosebaceous follicles. It is mainly composed of macromolecules, fibroblast cells, and immune system cells. The epidermis is the thinnest surface of the skin. It is composed of 90% keratinocytes, which synthesize keratin and, thanks to their differentiation, ensure impermeability and protection for the skin.The epidermis is made up 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 spinosuni), composed of large keratinocytes that gradually flatten towards the upper layers; the granular layer (stratum granulosuni), composed of flattened keratinocytes that allow the keratinocytes to transform into comeocytes (anucleated cells) and finally the horny layer (stratum corneum), the upper layer composed of comeocytes that will desquamate. The epidermis is not only composed of comeocytes but also of lipids between these different cells. Lipids are organized in the form of lipid bilayers. The main classes of these lipids are ceramides, cholesterol and free fatty acids. They partly ensure the barrier function of the skin.

[0006] Sebum is a hydrolipidic film produced by the sebaceous glands. It is excreted on the surface of the skin 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 preserve lipophilic and anaerobic microbial species. It also protects the skin by lubricating and acidifying it to regulate skin pH.

[0007] The skin microbiota also plays a key role in the skin's protective functions. It also helps regulate the skin's pH. The skin microbiota, also called skin flora, is located in the epidermis. It is made up of microorganisms such as bacteria, viruses, fungi, and parasites. The skin microbiota evolves as we age, reaching an average of 1,000 billion bacteria and 1,000 species of viruses, parasites, and fungi. For bacteria, the most well-known and studied strains are Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. Some are aerobic and therefore live with 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 in some cases, even amino acids.Most of these bacteria use lipids from sebum to grow and produce these compounds.

[0008] The skin microbiota varies quantitatively and qualitatively from one person to another. Variables such as age, sex, immune system, pH, temperature, and humidity can alter 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: "resident" flora and "transient" flora.

[0009] As its name suggests, resident skin flora lives in complete symbiosis with the skin. We speak of commensal organisms. It is important for defense against pathogenic microorganisms, because the space it occupies saturates the area and prevents the attachment of unwanted organisms.

[0010] Transient flora does not establish itself permanently on the skin's surface; it varies throughout the day, depending on the activities carried out and changes in environmental conditions. It can be present for a few hours or a few days. The microorganisms that compose it are mostly harmless, called saprophytes. This flora can also be made up of opportunistic pathogenic bacteria and cause disease if the host's defenses weaken. One of the most common transient species is S. aureus, implicated in atopic dermatitis (AD).

[0011] The microorganisms present on the skin and composing the resident flora are not opportunistic; they fulfill 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 the homeostasis of the skin, by providing it with nutrients or by competing with pathogens. S. epidermidis, for example, secretes antimicrobial peptides against S. aureus. Corynebacterium striatum and C. acnes 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 particularly essential for good skin health. When this balance is disrupted, we speak of dysbiosis.It has been shown in numerous studies that dysbiosis is correlated with the appearance of certain inflammatory skin pathologies, such as acne, atopic dermatitis or eczema. Understanding the interactions between the skin microbiota and the skin therefore constitutes a challenge in the development of new cosmetic and pharmaceutical treatments.

[0012] Today, the evaluation of cosmetic and pharmacological agents can be carried out 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 corneum model from keratinocytes taken from the heels of volunteers (Acta Derm Venereol. 2016; 96(7): 873-879). A microbiota is notably added to the suspended keratinocytes. However, the composition of these skin models still differs greatly from the actual composition of human skin. In particular, it does not include lipids that have a protective role for the skin and the microbiota and that are secreted by the sebaceous glands. In addition, it does not contain viable keratinocytes.Thus, the interaction between microorganisms and keratinocytes (secretion of cytokines, antimicrobial peptides, etc.) is not observable.

[0013] 3D models of reconstructed epidermis with a microbial compound have also been used to study the interactions between the microbiota and the skin (Rademacher et al., Experimental Dermatology, 27, pp489-494, 2018). However, most of these models include the colonization of a single species of microorganism, such as C. amycolatum, and therefore do not reflect the real conditions of our skin. The presence of lipids in healthy skin models is also not described.

[0014] Therefore, there is still a need for a model that can reproduce the conditions closest to natural skin, preferably one that can be obtained easily and at low cost.

[0015] Advantageously, the inventors have developed a new skin model comprising a microbial component and a lipid component which advantageously makes it possible to approximate real skin. Advantageously, the cost associated with such a model is notably reduced compared to explants. Furthermore, this model can be used with different stresses in order to mimic skin disorders. Indeed, the inventors have notably observed that the application of exogenous stress on the skin model induced molecular and metabolic characteristics similar to those observed in vivo in subjects with skin exposed to the sun or to inflammatory conditions. The model can advantageously serve as a tool to screen active ingredients or even cosmetic or pharmacological formulations of interest.

[0016] Statement of the invention

[0017] The first object of the invention therefore concerns a reconstituted skin model comprising:

[0018] - a skin substitute, and

[0019] - a microbial component and a lipid component on the surface of said skin substitute.

[0020] Another object of the invention relates to a method for obtaining a reconstituted skin model comprising: a) Providing a skin substitute, b) Applying to said skin substitute, concomitantly or sequentially, a microbial component and a lipid component.

[0021] The invention also relates to a screening method for the in vitro identification of an active ingredient or a cosmetic formulation for preventing and / or improving at least one skin disorder induced following at least one exogenous stress comprising the following steps: a) Applying said active ingredient or said cosmetic formulation to the reconstituted skin model according to the invention; b) Carrying out at least one exogenous stress on said model; c) At least one measurement of the level of expression of at least one biological marker; and d) Determining whether said active ingredient or said cosmetic formulation makes it possible to prevent and / or improve at least one skin disorder based on at least one level of expression measured in step c).

[0022] The invention also relates to a method for evaluating the in vitro efficacy of an active ingredient or a 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 said cosmetic formulation to the reconstituted skin model according to the invention; b) Carrying out at least one exogenous stress on said model; c) At least one measurement of the level of expression of at least one biological marker; and d) Evaluating the efficacy of said active ingredient or said cosmetic formulation as a function of at least one level of expression measured in step c).

[0023] The invention also relates to a method for in vitro evaluation of the tolerance of an active ingredient or a cosmetic formulation, comprising the following steps: a) Application of said active ingredient or said cosmetic formulation to the reconstituted skin model according to the invention; 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. The invention finally 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.

[0024] Description of figures

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

[0026] Detailed description of the invention

[0027] As indicated above, the invention relates to a reconstructed skin model comprising:

[0028] - a skin substitute, and

[0029] - a microbial component and a lipid component on the surface of said skin substitute. By "skin substitute" is meant a skin cell culture composed of at least one layer. The skin substitutes according to the invention include in particular monolayer skin cell cultures, bilayer skin cell cultures and tissue models, including cultures of reconstructed epidermis or reconstructed skin. Indeed, as it is often difficult to work on fresh explants, it is particularly advantageous, in the context of the present invention, to use skin cell cultures. By "reconstructed epidermis" is meant an epidermis generated in vitro by 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):203-1 1).“Reconstructed skin” means 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, the skin cells include normal, healthy or pathological cells that can cause skin disorders, or cells from lines. Cultured skin cells can in particular be obtained from skin tissue explants. By "explant" or "skin explant" is meant here a sample of skin cells or tissue, which can be taken for surgical purposes or to carry out analyses. In particular, an explant can be obtained during a surgical excision. By "excision" is meant here a surgical procedure consisting of cutting (excising) a more or less wide or deep part of the skin to treat an anomaly or growth. An excision is carried out either to remove a cancerous tumor or one suspected of being cancerous, or to treat a benign skin anomaly 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 (mammoplasty, abdominal surgery, facelift, preputial sampling, otoplasty, i.e., ear reattachment, syndactyly or extra finger, etc.). An explant can also be obtained by biopsy. By "biopsy" is meant here a sample of skin cells or tissue taken for analysis purposes. Several types of biopsy procedures are known and practiced in the field. The most common types include (1) incisional biopsy, in which only a sample of the tissue is taken, (2) excisional biopsy (or surgical biopsy) which consists of the total removal of a tumor mass, thus performing a therapeutic and diagnostic procedure, and (3) needle biopsy, in which a sample of tissue is taken with a needle, which may be wide or thin.Other types of biopsy exist, such as smear or curettage, and are also encompassed by the present invention.

[0031] 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.

[0032] The skin cells according to the invention, whether originating from a biopsy or obtained by differentiation of stem cells, comprise at least one type of cell usually present in the hypodermis, the dermis and / or the epidermis. These cells thus comprise, 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.

[0033] Many methods for culturing skin cells are known to those skilled in the art. Any of these methods can be used to cultivate the skin substitute of the invention. The skin substitute is cultivated and / or stored under conditions that maintain, at least partially, cellular metabolism and / or cellular functions. The culture of the skin substitute therefore includes both monolayer skin cell cultures (e.g., keratinocytes) or bilayer skin cell cultures as well as tissue models, including reconstructed skin cultures.

[0034] Monolayer or bilayer skin cell cultures have been known and used for a very long time. Furthermore, many 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., EurJ 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., Altern Lab 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 Bl; EP 1 878 790 Bl; 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 those skilled in the art and are included within the scope of the invention.

[0035] Preferably, the reconstructed skin model is selected from the group comprising epidermis models consisting mainly of keratinocytes, skin models comprising a dermis and an epidermis, and skin models comprising a dermis, an epidermis and a hypodermis. The models comprising at least one dermis form connective tissues, while the models comprising at least one epidermis form stratified epithelia comprising the characteristic layers of the tissue considered. For example, it is possible to identify in the epidermis models a basal layer (stratum basalts), a spinous layer (stratum spinosum), a granular layer (stratum granulosum), and a horny layer (stratum corneum).

[0036] Advantageously, said skin model is an epidermis model comprising a matrix support preferably chosen from: an inert support chosen 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, polypropylene, polyethylene terephthalate (PET) membrane, a semi-permeable inorganic Anopore membrane, acetate or cellulose ester (HATF), a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane; in this group are the reconstructed Epidermis models (Skinethic®) as well as the EpiDerm® model, (Mattek Corporation); a film or membrane based on hyaluronic acid and / or collagen and / or fibronectin and / or fibrin.

[0037] In this group we can particularly mention the models: Laserskin® (Fidia Advanced Biopolymers), Episkin ® (L'Oréal).

[0038] These models can be seeded by fibroblasts in the dermal part.

[0039] These models, in which fibroblasts may or may not be integrated, 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.

[0040] There are also models dedicated to tissue therapy that can also be used in the context of the present invention. Examples include the Epidex (Modex Thérapeutiques), Epibase® (Laboratoire Genévrier), Epicell™ (Genzyme), Autoderm™ and Transderm™ (Innogenetics) models.

[0041] The matrix support is then seeded by keratinocytes to reconstruct the epidermis and ultimately obtain reconstructed skin.

[0042] Advantageously, the skin model used comprises 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 cutaneous appendages, such as hair, sebaceous glands.

[0043] The reconstructed skin model of the invention is particularly simple to implement. In addition, it does not require the use of a particular commercial cell line, and proves advantageously adaptable.

[0044] By "microbial component" is meant a set 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 set 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 the set of 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.

[0045] Preferably, the microbial component corresponds to the skin microbiota of at least one subject. By "microbiota" is meant the set of microorganisms that is present in an environment. The skin microbiota therefore corresponds to the set of microorganisms present on the skin. Of course, the composition of the microbiota can vary depending on the site of the skin. Preferably, the cutaneous microbial component corresponds to the microbiota of the facial skin, preferably the forehead, of at least one subject. Also, according to a preferred embodiment, the cutaneous microbial component comes 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 sample of the microbial component can be taken 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 10. 5 CFU / ml) at the time it is added to the surface of the skin substitute. After 48 hours of incubation, the microbial component preferably has a viability of approximately 100,000 RLU / 0.66 cm 2 skin substitute. Viability is determined using Promega's “BacTiter ATP Lite” kit.

[0046] 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 lipid component protects 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, C18:0 / C18:0, 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 30-50% w / w triglycerides. Preferably, the lipid component comprises 15-30% w / w free fatty acids. Preferably, the lipid component comprises 25-30% w / w waxes and / or wax esters. Preferably, the lipid component comprises 12-20% w / w squalene. Preferably, the lipid component comprises 4.5-8.5% w / w cholesterol and / or cholesterol esters. More preferably, the lipid component is composed of 30 to 50% w / w triglycerides, 15 to 30% w / w free fatty acids, 25 to 30% w / w waxes and / or wax esters, 12 to 20% w / w squalene and 4.5 to 8.5% w / w cholesterol and / or cholesterol esters. Preferably, the lipid component comprises 3.0 to 6.0% w / w cholesterol and 1.5 to 2.5% w / w cholesterol esters. Preferably, the lipid component is in hydrolipid form, i.e.in the form of an emulsion with water. Preferably, the lipid component is sebum. Preferably, the lipid component is obtained from a sample taken from 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 ug / cm. 2 , more preferably at a final concentration of 40ug / cm 2 of skin substitute. The skin substitute being composed of at least one layer of skin cells, the microbial component and the lipid component are present on the surface of said substitute.

[0047] By "subject" is meant here any human person, whether an adult or a child. By "child", according to the invention, is meant an individual whose age is less than or equal to 16 years. Thus, the category of children according to the invention includes newborns, whose age is between 0 and 1 month, infants, who are between 1 month and 2 years, and children proper, who are at least 2 years old. A "newborn", as used here, can be born at term or premature. An "adult" within the meaning of the present invention is a person who is not a child, in other words a person over 16 years old.

[0048] Another object of the invention relates to a method for obtaining a reconstituted skin model comprising: a) Providing a skin substitute, b) Applying to said skin substitute, concomitantly or sequentially, a microbial component and a lipid component.

[0049] The skin substitute is as described above. The lipid component may be applied before the microbial component, or vice versa. Preferably, the microbial component and the lipid component are mixed together before application to the skin substitute. Preferably, the microbial component and / or the lipid component is / 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.

[0050] According to a particular aspect, the reconstituted skin is stressed. Preferably, the method of obtaining 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 c) of carrying out at least one exogenous stress.

[0051] Step c) may be carried out before or after step b) of the method for obtaining the model. In some cases, step c) may be carried out on the microbial component and / or the lipid component before their application to the skin substitute. Preferably, step c) is carried out after step b) of the method.

[0052] Exogenous stress refers to any external factor that affects the integrity of the skin and may lead, if necessary, to a progressive reduction in the effectiveness of its functions. Exogenous stress can be chemical and / or physical.

[0053] Physical stresses include environmental stresses (e.g. temperature) and mechanical stresses (e.g. friction). Preferably, the physical stress is selected from ultraviolet radiation, sunlight, infrared, near infrared, thermal radiation, radio waves including microwaves and mobile phone waves, ionizing radiation including beta, gamma, X, non-ionizing radiation, radiation from a magnetic field, ozone, a change in pressure, heat, cold, friction, stretching, particles.

[0054] 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 visible blue light and / or infrared. By "ultraviolet radiation" is meant electromagnetic rays whose wavelength is from 153 nm to 400 nm. Preferably, the exposure to UV according to the invention comprises exposure to UVA and / or UVB. By UVA, for the purposes of the invention, is meant radiation whose wavelength is from 400 to 320 nm. By UVB, for the purposes of the invention, is meant radiation whose wavelength is from 320 to 290 nm. By "visible light" is meant radiation whose wavelength is from 400 to 700 nm. "High energy visible blue light" means visible light with a wavelength between 400 and 450 nm.By "infrared" we mean radiation with a wavelength between 700 nm and 2500 nm.

[0055] According to a preferred embodiment, the UV exposure according to the invention comprises or consists of exposure to UVA. According to another preferred embodiment, the UV exposure according to the invention comprises or consists of exposure to UVB. According to an advantageous embodiment, the UV exposure according to the invention comprises or consists of exposure to UVA and UVB. According to another advantageous embodiment, the radiation exposure according to the invention comprises or consists of exposure to UVA and / or UVB and / or visible high-energy blue light and / or IR. In a 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 / cm 2for approximately 45 minutes. In another particular embodiment, the UVB dose is between 80 and 150 mJ / cm 2 UVB; preferably 120 mJ / cm 2 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).

[0056] Preferably, the chemical stress is at least one chemical substance whose topical application leads to an alteration of the structure and / or function of the skin. Preferably, the chemical stress is at least one allergen, a pollutant, a surfactant, a solvent, or at least one pro-inflammatory stimulation reagent such as a cytokine; preferably a cocktail of specific cytokines capable of inducing the skin disorder studied. Such reagents are well known, and are described for example in application WO 2015 / 014949. According to a particular aspect, the chemical stress comprises one or two components chosen from interleukin 1b, a ligand of the TLR-2 receptor, and a ligand of the TLR-3 receptor. Preferably, the chemical stress comprises interleukin 1b and optionally the TLR ligand (Poly (1:C) and / or Pam3CSK4, more preferably all three components.The implementation of a chemical stress comprising the application of at least interleukin IL-1P on the reconstituted skin model advantageously makes it possible to mimic AD. Of course, other chemical stresses can also make it possible to mimic AD, such as the combination of Poly I:C and TNFa; the combination IL4 and IL13; the combination Poly LC, IL-4, IL13; the combination IL-4, IL-13, IL-31 and optionally TNFa; the combination IL-4, IL-13, IL-22, TNFa.

[0057] As a 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, "particles matter" (PM10), from batches ERM-CZ100 / CZ120, are in the form of very fine dust. For example, 10 uL / cm 2A solution of fine particles suspended in acetone can be deposited on the surface of the reconstructed epidermis before adding the 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 representative of outdoor pollutants because it leads to highly toxic metabolites. Alternatively, Tetrabisphenol A (TBBPA), which is a brominated flame retardant found in most indoor 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. Persistently high levels of cortisol can be very damaging to the skin. Negative effects can include unwanted visible signs of aging such as fine lines and wrinkles, thinning of the skin, reduced elasticity, and reduced functionality of the skin barrier.In order to obtain a stressed skin model, step (c) may correspond to bringing the skin substitute into contact with cortisol, for example at a concentration of 4 pM.

[0058] Without being limiting, certain exogenous stresses, making it possible 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.

[0059] [Table 1]

[0060]

[0061] The invention also relates to a reconstructed skin model obtained by the method as described herein.

[0062] In a particular embodiment, at least two exogenous stresses, identical or different, are carried out on the reconstituted skin model according to the invention.

[0063] In another aspect, the invention makes it possible to identify active ingredients or formulations for preventing and / or improving at least one skin disorder or to evaluate the effectiveness of active ingredients or formulations for preventing and / or improving at least one skin disorder. The invention makes it possible in particular to distinguish the active ingredients or formulations according to their activity for preventing and / or improving at least one skin disorder.

[0064] The invention therefore also relates to a screening method for the in vitro identification of an active ingredient or a cosmetic formulation for preventing and / or improving at least one skin disorder induced following at least one exogenous stress comprising the following steps: a) The application of said active ingredient or said cosmetic formulation to the reconstituted skin model; b) Carrying out at least one exogenous stress on said model; c) At least one measurement of the level of expression of at least one biological marker; and d) Determining whether said active ingredient or said cosmetic formulation makes it possible to prevent and / or improve at least one skin disorder based on at least one level of expression measured in step c).

[0065] The invention also relates to a method for evaluating the in vitro efficacy of an active ingredient or a 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 said cosmetic formulation to the reconstituted skin model; b) Carrying out at least one exogenous stress on said model; c) At least one measurement of the level of expression of at least one biological marker; and d) Evaluating the efficacy of said active ingredient or said cosmetic formulation as a function of at least one level of expression measured in step c).

[0066] Step a) may be carried out before or after step b) of the screening method for the in vitro identification of an active ingredient or a cosmetic formulation or in the method for evaluating the in vitro efficacy of an active ingredient or a cosmetic formulation. Indeed, carrying out step a) before step b) may in particular make it possible to evaluate the capacity of an active ingredient or cosmetic formulation to prevent at least one skin disorder, whereas carrying out step a) after step b) may in particular make it possible to evaluate the capacity of an active ingredient or cosmetic formulation to improve at least one skin disorder.

[0067] The application of the active ingredient of interest to the skin model according to step a) can be done directly. Alternatively, it may be advantageous to formulate the active ingredient of interest, for example so as to obtain a liquid composition, in order to facilitate its application to the skin model. Thus, according to one embodiment of the invention, the method further comprises a step of formulating the active ingredient, in particular in the form of a liquid solution, in particular aqueous, prior to step a) of applying said active ingredient with a skin model.

[0068] 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 makes it possible to modulate 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 makes it possible to modulate 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.

[0069] For example, it would be beneficial to identify actives 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.

[0070] For the purposes of this application, the term "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 cancel or reduce the effects associated with at least one skin disorder. Prevention in this case means a treatment that is administered before the development of the skin disorder (i.e. before the occurrence of exogenous stress in step b)), while reduction corresponds to a treatment that is administered once the effects of the skin disorder have appeared (i.e. after the occurrence of exogenous stress in step b)).

[0071] The term "increased," as used herein, means a greater quantity, for example, a quantity slightly greater than the original quantity, or for example, a quantity in great excess of the original quantity, and especially all quantities in the interval.Alternatively, "increase" may refer to an amount 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 amount or activity to which the increased amount or activity is 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%, 650%, 700%, 750%, 800%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900% or 2000% greater than the quantity or activity to which the increased quantity or activity is compared. The terms "increased", "greater than", and "increased" are used interchangeably herein.

[0072] The term "diminished" as used herein means a lesser quantity, for example, a quantity slightly less than the original quantity, or for example a quantity greatly reduced from the original quantity, and including all quantities in the interval.Alternatively, "decrease" may refer to an amount 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 amount or activity to which the decreased amount or activity is 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%, 650%, 700%, 750%, 800%, 900%, 950%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900% or 2000% less than the quantity or activity to which the diminished quantity or activity is compared. The terms "diminished", "less than", and "reduced" are used interchangeably herein.

[0073] 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.

[0074] According to this preferred embodiment, step d), making it possible to determine whether the active ingredient or the cosmetic formulation prevents and / or improves at least one skin disorder, may comprise a comparison of the level of expression of the biological marker from step c) with a reference level of expression.

[0075] For the purposes of the present application, the term "a reference expression level of a biological marker" means any level of expression of said marker used as a reference. For example, a reference expression level may be obtained by measuring the level of expression of the marker of interest in a normal skin model. Such a normal skin model may, for example, correspond to a reconstituted skin model obtained from skin cells of a healthy subject or one without apparent pathology. A reconstituted skin model without the occurrence 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 said biological marker.

[0076] 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 a particular embodiment, the reference expression level of a biological marker is obtained by measuring the expression of said marker in the skin model which has not been contacted with an active ingredient or a formulation and / or which has not undergone exogenous stress. In another particular embodiment, the expression of said marker is measured in the skin model treated with an active ingredient or a formulation known to be effective against a skin disorder.

[0077] The person skilled in the art will furthermore readily understand that the comparison of step d) is preferably carried out between measurements of expression levels obtained for skin models obtained from skin substitutes of similar, or even identical, histological structures. For the purposes of the present application, the term "similar histological structures" means that the relative proportions of the cell types included in the skin models compared are similar. Thus, it is preferable that the relative proportions of the cell types and / or types of microorganisms included in the skin model of step a) do not differ by more than 5% from the relative proportions of the cell types and / or types of microorganisms included in the skin model used to obtain the reference expression level of step d).For the purposes of the present application, the term "relative proportion of a cell type" means the ratio of the number of cells corresponding to this cell type to the total number of cells included in the skin model. Thus, it is for example preferable that the proportion of keratinocytes to the total number of cells in the skin model of step a) does 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 of step d). For the purposes of the present application, the term "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 included in the skin model. Preferably, the relative proportion of a genus or species of bacteria is the ratio of the abundance of said genus or species to the total number of bacteria.

[0078] For the purposes of the present application, the term "identical histological structures" means that the relative proportions of the cell types included in the compared skin models are identical. For the purposes of the present 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 out of 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 out of the total number of cells in the skin model used to obtain the reference expression level of step d).

[0079] The person skilled in the art will also readily understand that the comparison of step d) is preferably carried out between measurements of expression levels obtained for skin models which are of similar, or even identical, size, volume or weight. Thus, it is preferable that the size, volume, or weight of the skin model of step a) does not differ by more than 5% from the size, volume, or weight of the skin model used to obtain the reference expression level of step d). More preferably, the size, volume, and weight of the skin model of step a) do not differ by more than 5% from the size, volume, and weight of the skin model used to obtain the reference expression level of step d).Even more preferably, the size, volume and weight of the skin model of 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 of step d).

[0080] Alternatively, if the skin models differ by more than 5% in size, volume and weight, the person skilled in the art may normalize the level obtained in step c) and the reference level of step d) using a normalization factor.

[0081] 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.

[0082] 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 almost 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 a normalization factor. According to another embodiment, the level obtained in step c) and the reference level of 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, which 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.

[0083] Step c) is carried out after steps a) and b). The determination of step d) will advantageously be carried out by comparing the level of expression of step c) with a reference level of expression (see above). For example, the skin model which has not been treated with the active ingredient or the formulation can be used as a control. In this case, the level of expression of the biological marker of the invention is measured in said skin model before and after application of the active ingredient or the cosmetic formulation. Alternatively, the level of expression of the biological marker of the invention is measured in a skin model without application of the active ingredient or the cosmetic formulation and in a skin model with the active ingredient or the cosmetic formulation. The level of expression of the biological marker of the invention can also be compared with that measured in vivo in normal skin or in vivo in skin having undergone exogenous stress.By "normal skin" is meant here skin from a healthy subject or without apparent skin pathology. The reference expression level is preferably the expression level of said biological marker in a skin model which has not been in contact with the active agent or the formulation, which allows a meaningful comparison to be made between the expression level of step c) and said reference level. For example, a skin model which has not been treated with the active agent or the formulation (i.e. without carrying out step a) or before carrying out 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 the cosmetic formulation (or before and after carrying out step a)).

[0084] The method of the invention may further comprise a comparison of the cell viability in the skin model treated with the active ingredient or the formulation and in the control sample. In this case, the active ingredient or the cosmetic formulation is well tolerated by the skin if the cell viability of the sample is not affected by the presence of the active agent or the cosmetic formulation. According to another preferred embodiment, the method of the invention therefore comprises an additional step of determining the cell viability in the skin model treated with the active ingredient or the cosmetic formulation, determining the cell viability in the control sample and comparing the two.

[0085] Numerous tests for determining cell viability are available to the skilled person and are commonly used in cosmetology. Particular mention may be made of the MTT test, described for example in Mosman et al. (J Immunol Methods, 65(1-2): 55-63, 1983).

[0086] By "skin disorders" is meant any 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 pores of the skin, the sweat and sebaceous glands attached to it, and / or the microbiota.

[0087] The skin disorders according to the invention are more particularly manifested by lesions, which correspond to damaged or poor condition skin. Damaged skin includes, for example, sensitive reactive skin, dry skin (xerosis), skin damaged by the sun, by radiation, by cold, by stress or by 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 by wounds or burns.By skin disorder, we therefore mean disorders as diverse as scabies, angiomas (including tuberous, subcutaneous or flat), hemangiomas, infant acne, adolescent acne, ichthyosis (e.g. vulgaris, congenital, harlequin, etc.), rosacea, pruritus, etc. A skin disorder can also be caused or exacerbated by an external infection, for example of parasitic, viral, bacterial or fungal origin. Skin disorders include, in particular, warts, prurigo strophulus, scabies, pediculosis of the scalp, and mycoses. The latter are parasitic infections caused by the proliferation of microscopic fungi that are parasites in the body. Among the most common mycoses, we can cite candidiasis and pityrosporosis, which are caused by the proliferation of yeasts on the skin.Preferably, the skin disorder is chosen from disorders linked to aging, pollution, radiation; inflammatory dermatoses, preferably atopic dermatitis, acne, psoriasis; rosacea; pruritus; an alteration of the barrier function; reactive skin; xeroses.

[0088] For the purposes of this application, the term "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 therefore designates a whole range of various substances and parameters. For example, a biological marker may be a substance whose detection indicates a particular pathological state (for example the presence of C-reactive protein as a marker of an infection), or on the contrary a substance whose detection indicates a specific physiological state. Preferably, said biological marker is a biological marker of a skin disorder if its level of expression is different in healthy skin and in skin presenting the clinical characteristics of said skin disorder.More preferably, said marker characterizes a skin disorder if said marker is differentially expressed in a skin model in which exogenous stress has been carried out and the skin model not having undergone exogenous stress. This results in a ratio in step d) which is different from 1 when the measurement of the level of expression is compared between these two conditions.

[0089] The biological marker according to the invention is preferably a gene, the products of a gene such as its transcripts and the 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 the person skilled in the art for measuring the expression of said biological marker. In a particular embodiment, at least two, three, four, five, six, seven, eight, nine, or ten biomarkers are measured. Said biomarkers are discriminating, thus making it possible to screen active ingredients or cosmetic formulations to prevent or treat skin disorders or to evaluate the effectiveness of active ingredients or cosmetic formulations to prevent or treat skin disorders.

[0090] A person skilled in the art seeking to determine to which class a biological marker belongs can easily consult the relevant scientific literature or refer to public databases such as, for example, those gathered on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0091] The biological marker of the invention is advantageously a marker selected from 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 component / lipid component which are present on the surface of said skin substitute of the model. According to one aspect, the biological marker is measured at least in the microbial component, preferably in the microbial component. According to another aspect, the biological marker is measured at least in the skin substitute, preferably in the skin substitute.

[0092] The "inflammatory 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.

[0093] Among cytokines, IL-8 protein is a cytokine that is induced early in cutaneous inflammation. It is notably produced by de novo synthesis in keratinocytes stimulated by IL1a 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 accessible under the NCBI reference: NM_000584.

[0094] TSLP is an inflammatory cytokine characteristic of AD. Its secretion is markedly stimulated in atopic dermatitis and promotes the initiation of an inflammatory response by Th2. 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.

[0095] The "oxidative stress markers" according to the invention comprise markers that are expressed in the skin and / or in the microbial component in the event of oxidative stress. Preferably, the oxidative stress markers are metabolites. Preferably, at least one metabolite from xanthine, uric acid, and glutathione is measured.

[0096] The "barrier function markers" according to the invention comprise markers which are specifically expressed in the outermost layers of the epidermis and which participate in the barrier function. Preferably, the barrier function markers according to the invention are markers expressed in the stratum corneum or markers expressed in the tight junctions of the stratum granulosum. These markers include claudins, including Claudin-1 (CLDN1), transglutaminases, such as transglutaminase 1 (TGM1), 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), sphingomyelinase or sphingomyelin diesterase (SMPD), comeodesmosin (CSDN), etc.Preferably, at least one marker of claudin-1 (CLDN1), transglutaminase 1 (TGM1), and filaggrin (FLG) is measured. In another aspect, the barrier function markers comprise one or more discriminating metabolites of barrier function. Preferably, at least one metabolite of octanoic acid, serine, glutamate, phosphorylcholine, histidine, L-glutamine, L-alanine, methionine, and xanthine is measured.

[0097] The "markers of the microbial component" according to the invention comprise markers which are modulated in the microbial component in response to an exogenous stress and / or an active ingredient or cosmetic formulation. They include in particular metabolites, such as formate, acetate, glucose and maltose, glycerol, betaine, octanoate, tyrosine, panthenol, phenylalanine. The expression of a gene 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 resulting from said transcripts. Thus, by "measuring the level of expression of a gene" is meant within the meaning of the invention the measurement of the quantity or cellular concentration of gene product in its peptide form or in its nucleotide form. In particular, the expression of at least one gene (e.g.16S) can be measured to determine the presence or absence of at least one microorganism in the model microbiota.

[0098] Generally, the expression of the biological marker according to the invention will be detected in vitro from the reconstituted skin model. In a particular embodiment, the method of the invention may comprise 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 directly used to measure the expression of the marker. The preparation or extraction of mRNA (as well as the reverse transcription thereof into cDNA), proteins, lipids or NMF from a skin cell sample are merely routine procedures well known to those skilled in the art.

[0099] In the case of markers that are secreted into the culture medium, the person skilled in the art can simply assay the marker from said culture medium.

[0100] Once a sample of mRNA (or corresponding cDNA) or protein is obtained, the expression of the marker, either at the mRNA level (i.e., in all mRNAs or cDNAs present in the sample) or at the protein level (i.e., in all proteins present in the sample), can be measured. The method used to do this then depends on the type of transformation (mRNA, cDNA or protein) and the type of sample available.

[0101] When the expression of the marker is measured at the level of mRNA (or corresponding cDNA), any technology usually used by the person skilled in the art can be implemented. These technologies for analyzing the level of gene expression, 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 nucleic acid chips (including DNA chips and oligonucleotide chips) for higher throughput.

[0102] Nucleic acid arrays herein mean several different nucleic acid probes that are 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.

[0103] The probes may be nucleic acids such as cDNAs (“cDNA chip”), mRNAs (“mRNA chip”) or oligonucleotides (“oligonucleotide chip”), said oligonucleotides typically being between about 25 and 60 nucleotides in length.

[0104] To determine the expression profile of a particular gene, a nucleic acid corresponding to all or part of said gene is labeled, then brought into contact with the chip under hybridization conditions, leading to the formation of complexes between said labeled target nucleic acid and the probes attached to the surface of the chip which are complementary to this nucleic acid. The presence of labeled hybridized complexes is then detected.

[0105] These technologies make it possible to monitor the expression level of a particular gene or 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 there is therefore no need to detail them here. Examples of implementations of the invention based on gene expression analysis (cDNA chips) and quantitative PCR are described in the experimental section.

[0106] Alternatively, any current or future technology that can determine gene expression based on the amount of mRNA in the sample can be used. For example, the skilled person can measure gene expression by hybridization with a labeled nucleic acid probe, such as Northern blotting (for mRNA) or Southern blotting (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. Assays commonly used with tissue microarrays include immunohistochemistry and fluorescent in situ hybridization. For analysis at the mRNA level, tissue microarrays can be coupled with fluorescent in situ hybridization.Finally, it is possible to use massive parallel sequencing to determine the amount of mRNA in the sample (RNA-Seq or "Whole Transcriptome Shotgun Sequencing"). For this purpose, several massive parallel sequencing methods 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.

[0107] When marker expression is measured at the protein level, specific antibodies can be used, particularly in well-known technologies such as immunoprecipitation, immunohistologic, western blot, dot blot, ELISA or ELISPOT, protein microarrays, antibody microarrays, or tissue microarrays coupled with immunohistochemistry.Other techniques that may 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 (voltammetry and amperometry techniques), and radiofrequency methods, such as confocal and non-confocal multipole resonance spectroscopy, such as the detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., 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 mass spectrometry and tandem mass spectrometry, by liquid or gas chromatography coupled with mass spectrometry or tandem mass spectrometry. All these techniques are well known to the person skilled in the art and it is not necessary to detail them here.

[0108] If the biological marker is a lipid, in particular a ceramide or a fatty acid, or a metabolite, the person skilled in the art may use all methods at his disposal to measure the lipid content in a sample of skin cells. These methods include, among others, liquid chromatography (HPLC, see for example Sullivan et al., Arch Ophthalmol., 120(12): 1689-99, 2002), liquid chromatography coupled with an evaporative light diffraction detector (HPLC-ESD, see Nordbâck 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-mass spectrometry (GC-MS, see O'Neill et al., J Chromatogr Sci., 14(1): 28-36, 1976); gas chromatography-flame ionization detector; liquid chromatography-mass spectrometry (see e.g. van Smeden et al., J Lipid Res, 52(6): 1211-1221, 2011); ultra-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 particularly 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 in electron microscopy (Daehnhardt-Pfeiffer et al., Skin Pharmacol Physiol., 25(3): 155-161, 2012) or by electron microscopy analysis of vitreous section of skin combined with molecular analysis (Iwai et al., J Invest Dermatol., Apr 26, 2012).

[0109] The NMF assay is a procedure well known to those skilled in the art. In particular, it is possible to assay NMF using in vivo confocal Raman microspectroscopy. This is a procedure that has been commonly used in the field for at least 15 years. Examples include the publications of Caspers et al. (J Invest Dermatol., 116(3): 434-442, 2001), Vyumvuhore et al. (J Biomed Opt., 19(11): 111603, 2014), or Falcone et al. (Skin Pharmacol Physiol, 28: 307-317, 2015). It is also possible to assay NMF using liquid chromatography coupled with mass spectrometry. Reference is made, for example, to Piraud et al. (Rapid Commun 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 even Yang (Application of biophysics and bioengineering to the assessment of skin barrier function.Thesis (Doctor of Philosophy (PhD)). University of Bath, UK, 2011).

[0110] In a particular embodiment, the active ingredient being evaluated is at least one sunscreen. A sunscreen is any substance capable of filtering solar radiation to protect a surface, typically the skin or hair, against the harmful effects of this radiation.

[0111] The sunscreen is chosen from those conventionally known including organic sunscreens such as triazine derivatives, benzotriazole and phenyl benzotriazole derivatives, dibenzoylmethane derivatives, amino-substituted benzophenone and 2-hydroxybenzophenone derivatives, merocyanine derivatives including methoxypropylamino cyclohexenylidene ethoxyethylcyanoacetate, para-aminobenzoic acid derivatives, salicylic derivatives, cinnamic derivatives, P,P'-diphenylacrylate derivatives, benzylidene camphor derivatives, phenylbenzimidazole derivatives, anthranilic derivatives, imidazoline derivatives, and benzalmalonate derivatives. inorganic sunscreens such as coated or uncoated metal oxides such as, for example, titanium, iron, zinc, zirconium, or cerium oxides.

[0112] Preferably, the active ingredient evaluated in is at least one sunscreen. Preferably, the method for evaluating the in vitro efficacy of at least one sunscreen or of 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.

[0113] Method for in vitro evaluation of the tolerance of an active ingredient or a cosmetic formulation, comprising the following steps: a) Application of said active ingredient or said cosmetic formulation to the reconstituted skin model; 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. The active ingredient is an active ingredient that is well tolerated by the skin if said active ingredient does not significantly modulate the expression of the biological marker. Similarly, the cosmetic formulation is well tolerated if the expression of the biological marker is not significantly modulated by its addition to the reconstituted skin model.By "significant" is meant a result obtained by implementing a statistical test, such as a univariate Wilcoxon test, and having a p-value of less than 0.05, preferably a p-value of less than 0.01, more preferably a p-value of less than 0.001. The appropriate statistical tests are well known to those skilled in the art. Said 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. In particular, the expression of inflammation markers is known to be increased when the skin is attacked, for example by exogenous stress. On the other hand, the expression of these inflammation markers is not affected by well-tolerated active ingredients or formulations. In a preferred embodiment, the biological marker of step b) is an inflammation marker, for example among those described here.

[0114] Preferably, the tolerance consists of maintaining the balance of the microbial component. By "maintaining the balance" is meant that at least one microorganism, preferably at least one bacterial genus or species, is maintained in the microbial component of the model following application of the agent or cosmetic formulation in step a). Preferably, the abundance of said at least one microorganism is not significantly modified following application of the agent or cosmetic 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.

[0115] Another subject of the invention is 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.

[0116] 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 the set of genera described above. Preferably, the at least four genera comprises the following genera: Cutibacterium, Staphylococcus, Streptococcus, and Burkholderia. The microbial component may also comprise one or more genera or species of fungi and / or viruses.

[0117] Preferably, the microbial component corresponds to the skin microbiota of at least one subject. By "microbiota" is meant the set of microorganisms that is present in an environment. The skin microbiota therefore corresponds to the set of microorganisms present on the skin. Of course, the composition of the microbiota can vary depending on the site of the skin. Preferably, the cutaneous microbial component corresponds to the microbiota of the facial skin, preferably the forehead, of at least one subject. Also, according to a preferred embodiment, the cutaneous microbial component comes from a sample of at least one subject, more preferably at the level of the face and even more preferably at the level of the forehead. Preferably, the microbial component has a viability of 5,000 RLU.

[0118] The lipid component is as described above.

[0119] Preferably, the kit further includes an instruction leaflet.

[0120] Preferably, the kit further comprises at least one chemical component as described herein. Preferably, the kit comprises an allergen, a polluting agent, a surfactant, a solvent, or at least one pro-inflammatory stimulating reagent such as a cytokine, or cortisol. Said kit advantageously makes it possible to obtain the reconstituted skin model. EXAMPLES

[0121] Example 1: Preparation of sebum and microbiota

[0122] Sebum preparation: Sebum is collected from the foreheads of 10 volunteers without apparent skin pathology using a standard collection kit. Each collection tube is then washed 3 times with hexane to extract the sebum. The extracts are then combined and the solvent is evaporated under vacuum. The mass of the collected sebum is controlled (e.g. 1,100 mg for one preparation) and the sebum is then taken up in 20 ml of hexane. 100 vials of sebum are prepared with 9 mg of substance per vial and packaged under argon. For the deposition of sebum on the reconstructed epidermis, the vials are then taken up with 250 μL of absolute ethanol (Fischer Scientific, Illkirch, France) and then diluted 1 / 20 eme in physiological saline. The sebum was characterized by GC / MS (Figure 1).

[0123] 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 rubbed vigorously on the donor's forehead for 4 seconds. Each swab was then placed in a tube for centrifugation. After centrifugation, a colorless liquid was collected (approximately 60 pL) and its volume was adjusted to 120 pL with physiological saline. Each microbiota was then mixed with the sebum in order to perform the deposition. 22 pL of the sebum / microbiota mixture was deposited on the surface of a reconstructed human epidermis and spread using a finger cot. A given sebum / microbiota mixture could be applied to up to 6 reconstructed epidermis. Human reconstructed epidermis are incubated at 32°C, 5% CO2, 60% humidity.

[0124] The incubation time is set at 48 hours, which allows for a sufficient quantity of bacteria without damaging the reconstructed human epidermis.

[0125] On the face, we find quantities of sebum ranging from >66pg / cm 2 for oily skin, 33-66pg / cm 2 , normal skin and <33pg / cm 2 for dry skin.

[0126] The inventors deposited on the reconstructed epidermis of 0.6 cm 2 , 24pg of sebum which corresponds to a concentration of 40pg / cm 2 .

[0127] 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, there are approximately 100,000 RLU per RHE of 0.6 cm 2, which corresponds to a growth of 1 to 2 log. Example 2: Establishment of the reconstructed skin model with or without the addition of stress Methods

[0128] The epidermis was reconstructed from skin excisions from cosmetic surgery according to the method described by Frankart et al. (Frankart et al., Exp. Dermatol. 2012, 21(11), 871-875).

[0129] Briefly, the cells (keratinocytes) were isolated from the skin excisions, then cultured before being seeded on culture inserts immersed in culture medium, then the culture inserts were placed at the air / liquid interface in an incubator at 37°C in a humidified atmosphere with 5% CO2, to form the stratum corneum.

[0130] At 14 days (D14), a reconstructed epidermis with a surface area of ​​0.6cm 2 is formed. The culture medium is renewed every 24 hours.

[0131] At day 10, the reconstructed epidermis were placed in a culture medium without antibiotics. At day 13, the reconstructed epidermis were placed in 12-well plates with maintenance medium without antibiotics (see Frankart et al., 2012) and with Phenol Red. After 2 hours, a sebum / microbiota mixture, obtained as described in Example 1, was deposited on the surface of each human reconstructed epidermis and spread using a finger cot. The reconstructed human epidermis were seeded with a microbiota inoculum with a viability of approximately 5000 RLU.

[0132] Epidermis 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 renewed every 24 hours for up to 48 hours of incubation.

[0133] In order to mimic inflammatory stress of the atopic dermatitis type, the medium used is the maintenance medium (1 mL / well) supplemented with interleukin 10 at 0.06 ug / mL, TLR ligand (Poly (1:C) at 10 pg / ml, and Pam3CSK4 at 5 pg / ml).

[0134] Measuring microbiota viability

[0135] Upon receipt, the viability of the microbiota is measured using the BacTiter ATP Lite kit, Promega following the supplier's protocol. The viability of the microbiota after culture is assessed using this same kit. The viability measurement is a bioluminescent test involving luciferase. The amount of light emitted is proportional to the amount of ATP present in the sample and therefore to the number of living cells. In order to monitor bacterial growth during the experiment, a viability reading is carried out in duplicate on the microbiota taken from each epidermis (BacTiter-Glo Microbial Viability Cell Assay kit, Promega). The ATP mix and the microbiota are placed in each well in a 1:1 ratio. After deposition, the plate (Low Binding Greiner 655209) is shaken for 30 seconds at 500 rpm then incubated at room temperature for 5 minutes. The reading is then carried out using a Clariostar plate reader.

[0136] Extraction of DNA from the microbiota

[0137] The DNA of microorganisms before and / or after growth on reconstructed epidermis is extracted using a Qiacube following the Qiagen QIAamp DNA Investigator Kit protocol.

[0138] Absolute quantification of microorganisms of interest in ddPCR

[0139] Droplet Digital PCR (ddPCR) is a digital PCR technique based on the generation of droplets using a water / oil emulsion. From a sample, it can generate more than 20,000 droplets within which PCR amplification is carried out. 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.

[0140] ddPCR is performed in a 96-well plate: 5 pL of sample is added to 15 pL of ddPCR mix as explained in the Biorad protocol. The probes used correspond to the species being tested for, e.g. S. epidermidis, S. aureus, C. acnes.

[0141] Droplet generation is performed using Biorad's QX200 Droplet Generator, and the plate is then placed in the thermal cycler for amplification. The plate is then read using Biorad's QX200 Droplet Reader and analyzed using QuantaSoft software.

[0142] Targeted sequencing

[0143] The impact of “culture on reconstructed human epidermis” on the microbiota is evaluated by targeted sequencing.

[0144] Sequencing was performed on a MiSeq sequencer (Illumina) with primers adapted to each target. Sequence preprocessing was performed using a computer pipeline developed by INRAE ​​running under Mothur (version 1.44.0).

[0145] 16S bacterial sequencing

[0146] The primers used target the variable regions VI -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). Barcodes and primers as well as chimeras were removed from the sequence files during the filtering steps. Sequences showing 100% homology between them were grouped into unique sequences, then into OTUs (operational taxonomy units: threshold of 97%) which will be identified subsequently.

[0147] Bioinformatics 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)). The analysis allowing phylogenetic affiliation down to the genus level was performed using bioinformatics tools for processing large amounts of sequence data (Mothur). Identification was performed based on the Greengenes taxonomy for bacteria.

[0148] Sequencing of target Staphylococcus species “Tuf”

[0149] The primers used target the TUF gene. Bioinformatic analysis of the sequencing data allows the identification of Staphylococcus present at different taxonomic levels (genus and species are targeted). The analysis allowing phylogenetic affiliation down to the species level was carried out using bioinformatic tools for processing large amounts of sequence data (Mothur). Identification was carried out on the Staphylococcus list.

[0150] Evaluation of the impact of cultured microbiota on healthy and / or stressed reconstructed epidermis

[0151] The expression of genes encoding barrier function proteins (Claudin 1, Filaggrin and TGM1) or human antimicrobial peptides (DEFB4A and S100A7) was assessed by RT-qPCR.

[0152] DNA extraction

[0153] The reconstructed epidermis halves are stored in 300 pL of a 1% β-mercaptoethanol solution of RLT (Qiagen) and frozen at -80°C pending extraction. First, the RHEs are separated from their membrane using forceps. To mechanically lyse the cells, a 5 mm Stainless Steel Beads (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. Secondly, RNA is extracted according to the Qiagen RNA Fibrous Tissue Mini Kit extraction protocol. Extractions are performed using Qiacube (Qiagen). The amount of extracted RNA is assayed using Nanodrop. RT-qPCR

[0154] The volume of RNA is then adjusted to reverse transcribe 1 pg of RNA per sample.

[0155] 4 pL of SuperScript VILO MasterMix is ​​then added to the samples.

[0156] The resulting cDNAs are then diluted 1 / 20 and arranged in 384-well plates (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. GAPDH, YWHAZ and UBC are the reference genes chosen for qPCR analysis.

[0157] IL-8 and TSLP assay by ELISA

[0158] IL-8 is measured in culture supernatants (24H, 48H and 72H) using the Bioplex ELISA IL-8 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 supernatants using the Bioplex TSLP kit from R&D Bio-Techne.

[0159] Results

[0160] Microbiota growth in the reconstructed skin model

[0161] Monitoring viability during the implementation time of this model accounts for the growth of microbiota on the epidermis.

[0162] The results of microbiota growth on healthy or inflammatory epidermis are presented in Table 2 below.

[0163] Growth of microbiota on epidermis assessed by the difference between viability at 48h - viability at T0. NS: non-significant; S: significant

[0164] [Table 2]

[0165] Microbiota growth on full epidermis is 1 to 2 log in 48 hours. Significantly greater growth is observed on inflammatory epidermis than on healthy epidermis. Without being bound by theory, it can be assumed that the barrier is altered in the inflamed epidermis, which allows microorganisms to access additional nutrients and therefore grow more.

[0166] Analysis of the microbiota component The bacterial composition of the microbiota was assessed at D13 (before inoculation on the model).

[0167] At D13, the bacterial genera identified in the microbiota are as follows:

[0168] Cutibacterium (more particularly C. acnes), Staphylococcus, Streptococcus, Burkholderia, Finegoldia, Gemella, Veillonella, Kocuia, Corynebacterium, Methylobacterium and Brevibacterium.

[0169] Staphylococcus species were further determined. Staphylococcus species identified in the microbiota were: S. epidermidis, S. capitis, S. warneri, S. hominis, and S. aureus.

[0170] Among the bacterial genera identified in the microbiota at 48 hours, Cutibacterium and Staphylococcus were notably detected. Staphylococcus species were determined more precisely. The Staphylococcus species identified in the microbiota at 48 hours are the same as those initially present (S. epidermidis, S. capitis, S. aureus, S. warneri, S. hominis).

[0171] Evaluation of the impact of microbiota / sebum on healthy epidermis

[0172] Effect of microbiota / sebum on the secretion of inflammatory cytokines by healthy epidermis 1 / Secretion of IL-8

[0173] The effects of microbiota / sebum on IL-8 secretion are summarized in Table 3.

[0174] IL-8 production kinetics as a function of microbiota / sebum growth time on epidermis.

[0175] [Table 3]

[0176] The presence of microbiota / sebum on healthy epidermis leads to the production of IL-8, so there is a reaction of the epidermis to the presence of microorganisms.

[0177] 2 / TSLP secretion TSLP is an inflammatory cytokine characteristic of atopic dermatitis. Its secretion is significantly stimulated in atopic dermatitis and promotes the initiation of an inflammatory response by Th2. It is measured in the supernatant after 24 hours of culture.

[0178] Healthy epidermis produces little TSLP.

[0179] The effects of microbiota / sebum on TSLP secretion are summarized in Table 4.

[0180] TSLP production by healthy epidermis with and without microbiota / sebum

[0181] [Table 4]

[0182] TSLP production is not modified by the presence of microbiota on healthy epidermis.

[0183] 3 / Effects on the expression of antimicrobial peptides

[0184] The DEEB4A gene encodes P-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.

[0185] The effects of microbiota / sebum on the expression of two antimicrobial peptides are shown in Table 5.

[0186] Expression of DEEB4A and S100A7 by healthy epidermis with and without microbiota

[0187] [Table 5]

[0188] RQ: “Relative quantification”, RQ > 2: overexpression, 0.5 < RQ: underexpression The presence of microbiota / sebum on healthy epidermis leads to a slight increase in the expression of the antimicrobial peptides DEFB4A and S100A7 by the epidermis. The presence of microbiota / sebum on healthy epidermis stimulates an “innate” immune response.

[0189] 4 / Effects on the expression of barrier function genes

[0190] 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 coding for Claudin-1, a protein involved in tight junctions, and the expression of the gene coding for Filaggrin. The TGM1 gene codes for transglutaminase-1, an enzyme involved in the formation of the stratum corneum and barrier function.

[0191] The effects of microbiota / sebum on TGM1 expression are presented in Table 6.

[0192] Expression of TGM1 by healthy epidermis with and without microbiota [Table 6]

[0193] RQ > 2: overexpression, 0.5 < RQ: underexpression

[0194] The presence of microbiota / sebum on healthy epidermis does not lead to significant changes in the expression of genes coding for proteins involved in barrier function, Filaggrin and Claudin 1 (results not shown). The expression of the gene coding for the enzyme TGM1 is increased.

[0195] Assessment of the impact of microbiota on inflammatory epidermis 1 / Secretion of IL-8

[0196] The effects of microbiota on IL-8 secretion are summarized in Table 7.

[0197] Illustrative example of IL-8 production at 24h and 48h on inflammatory epidermis [Table 7]

[0198] IL-8 production is increased 1.7 to 2.2 times. The presence of microbiota / sebum on inflammatory epidermis leads to a significant increase in IL-8 production, which reflects an amplification of the inflammatory reaction of the epidermis in the presence of microbiota.

[0199] 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).

[0200] 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.

[0201] 2 / Secretion of TSLP

[0202] The effects of microbiota / sebum on TSLP secretion are summarized in Table 8.

[0203] TSLP production by inflammatory epidermis with and without microbiota [Table 8]

[0204] The production of TSLP by inflammatory epidermis allows us to validate this model.

[0205] 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 modify TSLP production.

[0206] 3 / Effects on the expression of antimicrobial peptides

[0207] The effects of microbiota on the expression of two antimicrobial peptides are shown in Table 9.

[0208] Expression of DEFB4A and S100A7 by inflammatory epidermis with and without microbiota / sebum.

[0209] [Table 9] Expression relative to the control healthy epidermis without microbiota. RQ > 2: overexpression, 0.5 < RQ: underexpression

[0210] The presence of microbiota / sebum on inflammatory 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 inflammatory epidermis is observed in the presence of microbiota / sebum.

[0211] 4 / Effects on the expression of barrier function genes

[0212] The effects of microbiota / sebum on TGM1 expression are shown in Table 10. TGM1 expression by inflammatory epidermis with and without microbiota / sebum.

[0213] [Table 10]

[0214] Calculation of RQs relative to gene expression in healthy epidermis without microbiota. RQ > 2: overexpression, 0.5 < RQ: underexpression. The presence of microbiota on inflammatory epidermis leads to a significant increase in TGM1 expression by the epidermis, which confirms an exacerbated sensitivity of the inflammatory epidermis in the presence of microbiota.

[0215] In view of the above results, the inventors clearly highlight the interest of the reconstituted skin model comprising a skin substitute as well as a microbial component and a lipid component. Indeed, in non-pathological conditions, the presence of a microbial component and a lipid component induces the production of certain inflammatory markers such as, for example, IL-8, and a slight increase in the expression of antimicrobial peptides thus stimulating innate immunity. This suggests that the microbiota induces a basal level of inflammation under these conditions. Furthermore, under conditions of chemical, inflammatory stress, the microbiota induces an exacerbated inflammatory response, which leads to an impaired barrier function. Thus, in the presence of inflammatory chemical stress, the model advantageously reproduces an inflammation of the atopic dermatitis type.

[0216] This model is therefore perfectly suited to evaluating new active ingredients that would be potentially useful in the prevention or treatment of atopic dermatitis.

[0217] This model is also suitable for evaluating new active ingredients that would be potentially useful in the prevention or treatment of inflammatory dermatoses, such as psoriasis, acne, seborrheic dermatitis, chronic urticaria, eczema, rosacea.

[0218] Finally, this integrated model is particularly interesting for testing new active ingredients with putative activity in protecting, or even strengthening, the barrier function.

[0219] Example 3: Evaluation of the effect of stress mimicking sun exposure on the reconstructed skin model as well as the effect of a sunscreen

[0220] Physical stress is applied to the reconstructed skin model including microbiota and sebum. Specifically, the model is exposed to UV radiation 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 to study the protection of the latter on this integrated model.

[0221] Table 11: Formulation A

[0222] Formulation A

[0223] [Table 11]

[0224] Methods

[0225] The model described in Example 2 was used.

[0226] Eighteen reconstructed epidermis (corresponding to 3 donors with six reconstructed epidermis obtained per donor) with microbiota from 18 separate 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 microbiota / sebum, i.e. at D14.

[0227] The reconstructed epidermis are irradiated with simulated solar radiation 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. The irradiance in the UV spectra is approximately 70 W / m2 from 290 to 400 nm. The 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 air flow.

[0228] Formulation A is applied at a dose of 2 mg / cm 2 on the surface of the reconstructed skin model 2 hours before exposure to radiation. After irradiation with the total solar spectrum (up to 790nm), the model is kept in culture for 24 hours at 32°C and 60% humidity. At the end of the experiment, the surface of the reconstructed epidermis is washed twice with 100pl of physiological saline, and then dried with a cotton swab. The purpose of this step is to recover the skin microbiota as well as lipids (here sebum) for analysis separately from the skin cells.

[0229] Metabolomic approach

[0230] At the end of the study, the reconstructed epidermis were separated from the insert and weighed to standardize the results obtained with the metabolomic approach. The reconstructed epidermis were ground and 108 samples were extracted. Briefly, the samples were ground in 2 x ImL of acetonitrile / water 1 / 9 (v:v) with a fastprep using 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, then normalized in 220 pL of D2O. The samples were analyzed by NMR and then mass spectrometry.

[0231] 1H NMR spectra were 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 cryoprobe with inverse detection attached to a Cryoplatform (the pre-amplifier unit). 1H NMR spectra were acquired using the 1D NOESY experiment with pre-saturation for water suppression (noesyprld), with a mixing time of 100 ms.

[0232] After NMR analyses, samples were transferred from NMR tubes to UHPLC vials. Samples were centrifuged at 9000 g for 5 min. A volume of 10 pL was 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 was used: 0-30 min: 0% to 100% B, 30-34 min: 100% B. Separation was performed at 30°C with a Hypersil Gold C18 column (100 x 2.1 mm, 1.9 μm) from Thermo Scientific (Les Ulis, France).The following electrospray parameters were applied: capillary voltage of 0.5 kV, sample 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, sample 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 were acquired with a Synapt G2-Si mass spectrometer from Waters (Manchester, UK), between m / z 50 and 800 in sensitivity and centroid modes. Samples are analyzed randomly, and a QC sample corresponding to a pool of all samples is analyzed 11 times along the sequence.

[0233] Structural identifications of 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).

[0234] Data analysis

[0235] Statistical analysis and network mapping are performed by the MetaboHUB-MetaToul-AXIOM platform.

[0236] The data are normalized to allow quantification of signals detected in multiple samples.

[0237] Principal component analysis is first applied to check the validity of the acquisition, to detect potential outliers and internal clusters.

[0238] In a biological context, confounding factors are generally encountered: these factors add noise to the data corresponding to unwanted variability. Several sources of noise are possible: experimental, instrumental... 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. Orthogonal partial least squares discriminant analysis (O-PLS-DA) aims to eliminate this unwanted variation. A permutation test is performed to assess the robustness of PLS-DA models. The value of the Variable Importance on Projection (VIP) (threshold=l) was used to find discriminating features.

[0239] For each discriminating characteristic, a univariate Wilcoxon test and a false discovery rate (FDR) correction are performed to account for multiple testing (FDR-corrected p-value threshold = 0.05).

[0240] Multivariate analyses were performed using SIM CA vl5 software (Umetrics, Umeå, Sweden). The mixOmics package (Rohart et al., 2017) was used to perform multilevel analyses.

[0241] To create the metabolic network, INRAE ​​developed and used a freely available software package, Toxalim: MetExplore. This is a web server that offers the possibility to link metabolites identified in untargeted metabolomics experiments in the context of reconstructed genome-wide 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.

[0242] Results :

[0243] Pharmacological analysis of pathways and metabolites

[0244] 1 / oxidative stress: metabolomic approach

[0245] The following table reports the observed variations (Log 2 of variations) under the effect of UV irradiation for different metabolites discriminating oxidative stress in the epidermis of the reconstituted skin model.

[0246] [Table 12]

[0247] MI: Non-irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; ns: not significant

[0248] A significant modulation of these metabolites is observed after exposure to radiation, with a strong increase in xanthine and uric acid and a significant decrease in glutathione. These results are in agreement with in vivo studies on human volunteers (Randhawa et al, PLoS One. 2014; 9(3): e90367). These results show the validation of the model according to the invention in order to study sun exposure and predict the in vivo effect on human skin.

[0249] Furthermore, these results show a significant protective effect of a formulation comprising phenylene bis-diphenyltriazine (Formulation A) on oxidative stress due to sun exposure. Also, the model also makes it possible to identify or evaluate the in vitro efficacy of an active ingredient to prevent and / or treat at least one skin disorder induced following at least one exogenous stress such as UV irradiation.

[0250] 2 / Barrier function and hydration: metabolomic approach

[0251] 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.

[0252] [Table 13]

[0253] ''il: Not irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; Ns: not significant

[0254] These results demonstrate that the topical application of a formulation comprising phenylene bis-diphenyltriazine (Formulation A) can significantly cushion the imbalances induced by UV radiation, and thus preserve the barrier function and hydration of the skin.

[0255] 3 / Microbiota: metabolomic approach

[0256] 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. [Table 14]

[0257] MI: Non-irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; ns: not significant

[0258] UV irradiation induces significant changes in a large number of metabolites. These results also demonstrate that the topical application of a formulation containing phenylene bis-diphenyltriazine (Formulation A) significantly reduces the imbalances induced by UV radiation, and thus maintains the balance of the cutaneous microbial component. Phenylenes bis-diphenyltriazine therefore exhibits a true symbiotic action.

[0259] 4 / C. acne

[0260] C. acnes is a very abundant bacterial species in the microbiota of the model.

[0261] The inventors demonstrated a significantly increased abundance under the effect of irradiation (see Table 15).

[0262] [Table 15]

[0263] MI: Non-irradiated; IR: Irradiated; CTRL: Control; FA: Formulation A; ns: not significant

[0264] These results demonstrate that the topical application of a formulation comprising phenylene bis-diphenyltriazine (Formulation A) makes it possible to very significantly reduce the imbalances induced by UV radiation, and thus to protect the microbiota and maintain the balance of the cutaneous microbial component.

[0265] Using this model of reconstructed human epidermis with microbiota and sebum, the inventors identified a very precise metabolomic signature of skin exposed to UV radiation, mimicking sun exposure.

[0266] This integrated model is particularly interesting because it allows us to get closer to the conditions observed in vivo.

[0267] Furthermore, the inventors have shown not only that the effects of different stresses can be observed at the metabolomic level but also that Formulation A helps protect the skin ecosystem from sun exposure and preserve the symbiosis and homeostasis of the skin.

[0268] These results confirm the validity of the reconstructed skin model. These results also confirm the validity of the reconstructed skin model for use in methods of screening or evaluating the efficacy of cosmetic active ingredients to prevent or treat skin disorders, as well as in methods of evaluating the tolerance of an active ingredient or a cosmetic formulation. Very advantageously, the effect of an exogenous stress or an active ingredient could be determined on the basis of not only a single marker but several markers, allowing the establishment of a metabolomic signature, and demonstrating the pathophysiological relevance of the reconstructed skin model.

Claims

CLAIMS Reconstituted skin model including: - 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. 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. 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.A model according to any one of claims 1 to 3, characterized in that the lipid component is obtained from a sample taken from at least one subject, preferably from the face. A model according to any one of claims 1 to 4, characterized in that the microbial component is a cutaneous microbial component obtained from a sample taken from at least one subject, preferably from the face and even more preferably from the forehead. 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. 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. A process according to claim 7, characterized in that the exogenous stress is chemical or physical stress. A process 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. A process according to claim 8, characterized in that the chemical stress is a pro-inflammatory stimulant or cortisol.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).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).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. A method according to claim 12 for evaluating the in vitro efficacy of at least one sunscreen or cosmetic formulation for preventing and / or treating at least one skin disorder induced by at least one exogenous stress consisting of UV, infrared, or blue light irradiation. A method for evaluating the in vitro tolerability of an active ingredient or cosmetic formulation, 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) Measuring at least one expression level 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. A method according to claim 15, characterized in that tolerance consists of maintaining the equilibrium of the microbial component.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.

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