METHOD FOR ASSESSING THE EFFECTS OF DEHYDRATION ON CHILDREN'S SKIN

Description

[0001] The skin is a collection of cells and macromolecules grouped together to form a strong and flexible tissue, covering the entire body. It is made up of two joined layers: the epidermis and the dermis, to which subcutaneous tissues can be added.

[0002] The epidermis, whose main role is to protect the body, constitutes the outermost layer of the skin and ensures its impermeability and resistance. It can be divided into four distinct cell layers: a basal layer ( basal layer ) , a thorny layer ( thorny bed ), a granular layer ( granular layer ), and a stratum corneum ( stratum corneum ) . Although different cell types coexist in the epidermis, keratinocytes are by far the most numerous (90%). Their characteristic activity is the synthesis of keratins, fibrous proteins insoluble in water that represent 95% of the total proteins of the epidermis.

[0003] The skin's main function is to establish a protective barrier against environmental damage while allowing some exchanges between the internal and external environments. This barrier function is primarily ensured by the stratum corneum ( stratum corneumThis layer makes the skin impermeable and hydrophobic, thus protecting the dermis from massive water ingress. It is also the stratum corneum that resists chemical aggressions. It is composed of dead, anucleate cells called corneocytes, filled with keratins and other products such as lipids, fatty acids, and ceramides. The corneocytes are linked by specialized tight junctions called corneodesmosomes, forming a compact layer whose cohesion is further reinforced by a lipid cement. Beneath the stratum granulosum, the tight junctions within the stratum corneum also contribute to the skin's barrier function (see, for example, Hogan et al., J Allergy, 2012: 901940).

[0004] On a daily basis, the skin faces various attacks. It is exposed, for example, to chemical agents such as soap, as well as physical stressors like friction from clothing and sun exposure. The epidermis and epidermal appendages must therefore constantly renew themselves to keep the skin healthy. Stem cells make these maintenance and repair processes possible. More specifically, the regenerative capacity of the epidermis is conferred by adult stem cells, which allow for the regular replacement of differentiated cells shed during keratinization. Epidermal stem cells give rise to keratinocytes, which differentiate into corneocytes, which are exfoliated during the desquamation process. This process is particularly crucial for the maturation and maintenance of the skin barrier function.

[0005] The barrier function is particularly important, especially for limiting water loss from the epidermis.

[0006] Indeed, regardless of skin type, skin can experience temporary dehydration, which can be linked to extrinsic factors (wind, cold, sun, detergents, etc.) and / or intrinsic factors (psoriasis, eczema, aging, etc.). This lack of water leads to a decrease in the effectiveness of the skin barrier. A cascade of events, known as the "dry skin cycle," then begins. First, the decrease in barrier function leads to a further drop in water content and a worsening of the barrier. In turn, keratinocyte proliferation increases (hyperkeratosis), and moderate inflammatory changes are induced as the skin attempts to repair itself. This hyperproliferative inflammatory state is crucial in the dry skin cycle because it leads to abnormal differentiation and the production of poor-quality materials and structures.Finally, the reduced activity of desquamation enzymes leads to thickening and loss of hygroscopicity of the stratum corneum.

[0007] Adaptation to extrauterine life is a process that begins at birth and continues throughout the first year of life. The first few months of postnatal life constitute a period of structural and functional reorganization of the skin, allowing for physiological adaptation to the extrauterine environment. For example, the immaturity of the newborn's skin is highlighted by the difference in structure and molecular composition of the stratum corneumcompared to adults. These are incomplete and thus continue to develop during at least the first 12 months after birth (Chiou et al., Skin Pharmacol Physiol, 17: 57-66, 2004; Nikolovski et al., J Invest Dermatol, 128: 1728-1736, 2008; Stamatas et al., Pediatr Dermatol, 27: 125-131, 2010; Telofski et al., Dermatol Res Pract, 2012: 198789, 2012). Furthermore, the results of two recent clinical studies (Fluhr et al., Br J Dermatol, 166(3): 483-90, 2012 and Fluhr et al., Br J Dermatol, 2014, doi: 10.1111 / bjd.12880) suggest that infant skin exhibits a certain immaturity in its ability to retain water and regulate related mechanisms. Moreover, these studies have shown that the epidermal barrier develops structurally from birth to the age of two and is therefore not fully competent during this period.This helps to explain the fragility of the skin of infants and young children and its susceptibility to chemical, physical and microbial aggressions.

[0008] Furthermore, incomplete skin maturation can have significant clinical consequences. It is therefore important to allow the skin to build and develop correctly and harmoniously; otherwise, its functional and structural organization could be compromised. In this regard, preserving the barrier function and the epidermis's capacity for renewal is crucial.

[0009] Thus, the immaturity of the barrier and the mechanisms regulating hydration in the baby's skin could make it even more vulnerable to exogenous or endogenous factors involved in dry skin.

[0010] Therefore, there is still a need, by integrating knowledge about children's skin and the role of dehydration, to identify and characterize improved active ingredients and formulations to best care for children's dry skin.

[0011] WO2014 / 009566 describes a method for identifying molecular markers characterizing children's skin, as well as a method for evaluating tolerance and identifying active ingredients or formulations for children's skin. WO2014 / 170495 relates to methods for identifying active ingredients selected from C7 avocado sugars to prevent or treat skin barrier deficiencies in children's skin. Yokota et al., 2014 (Experimental Dermatology, vol. 23, 27-31) describe reconstructed human epidermal equivalents (Episkin™< Large Model RHEE) cultured under desiccation conditions induced by ampoules containing CaCl₂ granules placed on the cultures. DESCRIPTION

[0012] The inventors have shown that children's skin, and in particular that of infants, is especially sensitive to dehydration. Specifically, they have identified biological markers whose expression is altered in dry skin. Such markers are particularly advantageous because they allow researchers to track the skin's response under desiccation conditions.

[0013] The inventors have developed methods for evaluating effectiveness in vitroactive ingredients and formulations for preventing the effects of dehydration on children's skin, using a skin model specifically capable of reproducing the characteristics of children's skin, and in particular that of very young children such as infants. Previous art studies relied on the use of adult populations to analyze the skin's response to dehydration (De Benedetto et al., J Allergy Clin Immunol., 127(3): 773-786, 2011) or on reconstructed adult skin models (Yokota et al., Exp Dermatol., 23 Suppl 1:27-31, 2014). However, the properties of the skin change during the first years of life (see for example Fluhr et al., Exp Dermatol., 19(6): 483-492, 2010; Fluhr et al., Br J Dermatol, 166(3): 483-90, 2012 and Fluhr et al., Br J Dermatol., 2014, doi: 10.1111 / bjd.12880) and it is unlikely that it is possible to accurately determine the effects of dehydration on children's skin from adult skin samples.

[0014] However, the inventors developed reconstructed skin models from samples taken from children and were able to test the effect of dehydration on these models. They observed that the expression of certain biological markers was altered when these reconstructed children's skin models were cultured under desiccated conditions. Some markers, such as inflammation markers, were more strongly expressed, while the expression of others, such as stem cell markers or those related to barrier function, was decreased. Conversely, the variations in the expression of these markers were reduced, or even eliminated, when the models were treated with active ingredients or formulations known to treat or prevent dry skin. This result underscores the physiological relevance of these markers.This further reinforces the importance of using reconstructed skin models from children, rather than adults, to isolate such markers.

[0015] The invention is in particular as defined in the claims.

[0016] The invention relates in particular to a method for evaluating the in vitro efficacy of an active ingredient or formulation in preventing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) bring said active ingredient or formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) culture the reconstructed skin model from step a) under desiccated conditions; c) measure the level of expression of a combination of biological markers in the skin model from step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) evaluate the efficacy of said active ingredient or formulation according to the level of step c).

[0017] The invention also relates to a method for evaluating the in vitro efficacy of an active ingredient or formulation in reducing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) cultivate the reconstructed skin model under desiccating conditions, said model being obtained from a skin sample from a child; b) bring said active ingredient or formulation into contact with the reconstructed skin model from step a); c) measure the level of expression of a combination of biological markers in the skin model from step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) evaluate the efficacy of said active ingredient or formulation according to the level of step c).

[0018] The invention also relates to a method for identifying an active ingredient or formulation for preventing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) bring an active ingredient or candidate formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) culture the reconstructed skin model from step a) under desiccation conditions; c) measure the level of expression of a combination of biological markers in the skin model from step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) determine whether said active ingredient or candidate formulation is a formulation or active ingredient for the prevention of the effects of dehydration on children's skin according to the level of step c).

[0019] The invention also relates to a method for identifying an active ingredient or formulation for reducing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) cultivate the reconstructed skin model under desiccating conditions, said model being obtained from a skin sample from a child; b) bring an active ingredient or candidate formulation into contact with the skin model from step a); c) measure the level of expression of a combination of biological markers in the skin model from step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) determine whether said active ingredient or candidate formulation is a formulation or active ingredient for reducing the effects of dehydration on children's skin according to the level of step c).

[0020] The invention also relates to a method for evaluating the tolerance of an active ingredient or formulation on dehydrated children's skin, said method comprising the following steps: a) bring said active ingredient or formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) culture the reconstructed skin model from step a) under desiccating conditions in the presence of the active ingredient or formulation; c) measure the level of expression of a combination of biological markers in the skin model from step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) determine whether said active ingredient, active ingredient or formulation is well tolerated by child skin according to the level of step c).

[0021] By "dry skin," we mean skin that has suffered a loss of moisture. Dry skin is rough, flaky, and uncomfortable, characterized by a deficiency of water in the stratum corneum. Dry skin is also known as xerotic skin.

[0022] Dry skin can be caused by prolonged exposure to low humidity, increasing dehydration stress to such an extent that the normal hydration gradient of the stratum corneum is altered. In this regard, it has been shown that an atmospheric humidity level below 30% leads to dry skin (Sunwoo et al., J Physiol Anthropol, 25: 7-14, 2006). External conditions such as intense, dry cold, physical or chemical irritations, or sun exposure can trigger and / or perpetuate skin dehydration.

[0023] Dry skin can also develop as a result of physical or chemical changes in the skin, such as aging, leading to alterations in the normal moisture gradient. These changes can also be caused by pathological conditions, such as atopic dermatitis.

[0024] By "desiccation condition," we mean any condition leading to water loss from the skin. Desiccation conditions are therefore conditions that cause a normally hydrated child's skin model to become dry. This water loss can be caused by external conditions in which the atmospheric humidity level is lowered. It can also be caused by a direct alteration of the skin's barrier function, for example, by an alteration of the Natural Moisturizing Factors (NMF) or the lipid film of the stratum corneum. The inventors have shown that a child's skin model cultured under conditions in which the atmospheric humidity level is abruptly lowered exhibits physiological and histological characteristics entirely similar to those of dry child's skin.These dry skin models developed by the inventors are particularly advantageous, since they allow the phenotype of dry skin in children to be reproduced as observed. in life. These models are all the more advantageous because other models that cause water loss by altering the lipid layer do not mimic the characteristics of dry children's skin as well. in life. Thus, for example, desiccation conditions are conditions in which the humidity level in the atmosphere is lower than that of typical skin cell culture conditions. For example, the humidity level in the atmosphere is less than or equal to 50%. For example, it is less than or equal to 45%, 40%, 35%, 30%, or 25%. For example, it is less than or equal to 25%.

[0025] According to this invention, the term "child" means an individual under the age of 16. This includes newborns (aged 0 to 1 month), infants (aged 1 month to 2 years), and children (aged 2 years or older). A "newborn," as defined here, may be born at term or prematurely.

[0026] To avoid any ambiguity, the term "child" used here without further clarification should be understood in its broadest sense, that is, as referring to a person under 16 years of age. An "adult" is a person who is not a child, in other words, a person over 16 years of age.

[0027] Preferably, the method can be used regardless of the skin's ethnic or geographic origin, or its phototype. It can therefore be of Caucasian, African, Asian, South American, Melanesian, or other origin; it can also have a phototype I, II, III, IV, V, or VI, without affecting the procedure. Indeed, its purpose is to identify biological markers that characterize any skin type and depend only on the donor's age.

[0028] The methods of the invention are based on the use of a suitable skin model, reproducing children's skin, particularly dry children's skin, and on the use of biological markers whose expression is affected by dehydration in a specific way in children's skin. The invention thus makes it possible to precisely determine which active ingredients have a beneficial effect on preventing or treating the effects of skin dehydration. The methods of the invention are also suitable for evaluating the activity of formulations. The inventors were thus able to show that certain formulations were more effective than others in preventing and / or limiting the effects of dry skin, thereby demonstrating the usefulness of the approach taken.

[0029] A method for evaluating effectiveness is described here. in vitroof an active ingredient or formulation to prevent or treat the effects of dehydration of children's skin, said method including the determination of the level of expression and / or activation of at least one biological marker.

[0030] More specifically, the method described here preferably includes the following steps: a) bring said active ingredient or formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) culture the reconstructed skin model from step a) under desiccating conditions; c) measure the level of expression of at least one biological marker in the skin model from step b); and d) evaluate the efficacy of said active ingredient or formulation according to the level in step c).

[0031] For illustrative purposes, the reconstructed skin model is cultured under desiccated conditions in step b) in the presence of the active ingredient or formulation. The inventors have developed dry skin models cultured in a dry atmosphere that reproduce the phenotype of dry skin in children as observed in life. Thus, for example, desiccation conditions are cell culture conditions in which the atmospheric humidity is lowered compared to typical skin cell culture conditions. For example, the atmospheric humidity is 50% or less. For example, it is 45%, 40%, 35%, 30%, or 25% or less. In one particular example, it is 25% or less. In another example, the active ingredient or formulation is removed prior to exposing said reconstructed skin model to desiccation in said step b).

[0032] Furthermore, a method for evaluating effectiveness is also described here. in vitro of an active ingredient or formulation in reducing the effects of dehydration on children's skin, characterized in that said process comprises the following steps: a) cultivate under desiccated conditions a reconstructed skin model, said model being obtained from a skin sample from a child; b) bring said active ingredient or formulation into contact with the reconstructed skin model from step a); c) measure the level of expression of at least one biological marker in the skin model from step b); and d) evaluate the efficacy of said active ingredient or formulation according to the level in step c).

[0033] Those skilled in the art will readily understand that steps a) and b) can be performed simultaneously or sequentially, as required. In other words, the reconstructed skin model can be cultured under desiccated conditions in step b) in the presence of the active ingredient or formulation. Alternatively, the skin model can first be cultured under desiccated conditions and then brought into contact with the active ingredient or formulation.

[0034] The term "efficacy of a formulation or active ingredient in preventing or reducing the effects of dehydration in children's skin" refers to the ability of the formulation or active ingredient to counteract or reduce these effects. Prevention, in this context, refers to treatment administered before the effects of dehydration develop, while reduction refers to treatment administered once the effects of dehydration have appeared.

[0035] By way of illustration, the candidate formulation or active ingredient is effective in preventing, treating, or reducing the effects of dehydration in children's skin if said candidate formulation or active ingredient increases or maintains the expression levels of at least one biological marker of barrier function, and / or at least one biological marker preferentially expressed in stem cells, and / or at least one lipid, and / or at least one NMF. Alternatively or in combination, the candidate formulation or active ingredient is effective in preventing, treating, or reducing the effects of dehydration in children's skin if said candidate formulation or active ingredient decreases or maintains the expression levels of at least one biological marker of inflammation.

[0036] For illustrative purposes, desiccation conditions are cell culture conditions in which the atmospheric humidity is lower than that of typical skin cell culture conditions. For example, the atmospheric humidity is less than or equal to 50%. For example, it is less than or equal to 45%, 40%, 35%, 30%, or 25%. For example, it is less than or equal to 25%. In a preferred embodiment, the sample donor is more specifically a donor aged between 0 and 1 month, between 1 month and 2 years, or between 2 and 16 years. In other words, according to this embodiment, the sample donor is chosen from the group consisting of newborns, aged between 0 and 1 month, infants, aged between 1 month and 2 years, and children aged between 2 and 16 years.Preferably, the sample donor is a newborn or an infant.

[0037] As an illustration, the level of expression of said biological marker in step c) is compared to a reference level of expression.

[0038] It is important to verify that the active ingredients and formulations of the invention are well tolerated. For example, some currently marketed products could cause irritation if used regularly. Such an effect can only worsen a developing or existing condition of dry skin.

[0039] This document describes a method for evaluating the tolerance of an active ingredient or formulation when a child's skin is dry, said method comprising the following steps: a) bring said active ingredient or formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) culture the reconstructed skin model from step a) under desiccated conditions in the presence of the active ingredient or formulation; c) measure the level of expression of at least one biological marker in the skin model from step b); and d) determine whether said active ingredient, active ingredient or formulation is well tolerated by child skin according to the level of step c).

[0040] The process may, for example, also include a comparison of cell viability in the reconstructed skin model treated with the active ingredient, composition, or formulation and in the control reconstructed skin model, i.e., dehydrated skin that has not been treated with the active ingredient or formulation. In this case, the cosmetic active ingredient or formulation is well tolerated by the child's skin if the cell viability of the reconstructed skin model is not affected by the presence of the agent or formulation.

[0041] According to another example, the process therefore includes an additional step of determining cell viability in the dehydrated reconstructed skin model treated with the active ingredient or cosmetic formulation, determining cell viability in the control reconstructed skin model and comparing the two.

[0042] Numerous tests for determining cell viability are available to those skilled in the art and are commonly used in cosmetology. One example is the MTT test, described for instance in Mosman et al. (J Immunol Methods, 65(1-2): 55-63, 1983).

[0043] For example, the candidate formulation or active ingredient is considered well tolerated by children's skin if it increases or maintains the expression levels of at least one biological marker of barrier function, and / or at least one biological marker preferentially expressed in stem cells, and / or at least one lipid, and / or at least one NMF. Alternatively, or in combination, the candidate formulation or active ingredient is considered well tolerated by children's skin if it decreases or maintains the expression levels of at least one biological marker of inflammation.

[0044] In another example, desiccation conditions are cell culture conditions in which the atmospheric humidity is lower than that of typical skin cell culture conditions. For example, the atmospheric humidity is 50% or less. Alternatively, it could be 45%, 40%, 35%, 30%, or 25% or less. In one particular example, it is 25% or less.

[0045] In another example, the expression level of said biological marker from step c) is compared to a reference expression level.

[0046] In another aspect, the invention makes it possible to isolate formulations or active ingredients that have an effect in preventing the effects of dehydration in children's skin. As the experimental examples show, the invention makes it possible, in particular, to distinguish active ingredients or formulations according to their activity in preventing the effects of dehydration in children's skin. The invention is therefore particularly well-suited for identifying formulations or active ingredients appropriate for this very specific skin type.

[0047] Therefore, a method for identifying an active ingredient or formulation for preventing the effects of dehydration of children's skin is also described here, characterized in that said method comprises the following steps: a) bring an active ingredient or candidate formulation into contact with a reconstructed skin model, said model being obtained from a skin sample from a child; b) culture the skin model from step a) under desiccation conditions; c) measure the level of expression of at least one biological marker in the skin model from step b); and d) determine whether said active ingredient or candidate formulation is a formulation or active ingredient for the prevention of the effects of dehydration of children's skin according to the level of step c).

[0048] For example, the reconstructed skin model is cultured under desiccation conditions in step b) in the presence of the active ingredient or formulation. Alternatively, the active ingredient or formulation is removed prior to exposing the reconstructed skin model to desiccation in step b). In another example, desiccation conditions are cell culture conditions in which the atmospheric humidity is lower than that of typical skin cell culture conditions. For example, the atmospheric humidity is 50% or less. For example, it is 45%, 40%, 35%, 30%, or 25% or less. In one particular example, it is 25% or less.

[0049] Similarly, the method of the invention makes it possible to isolate active ingredients or formulations that reduce the effects of dehydration in children's skin. This method, comprising the following steps, is described herein: a) cultivate under desiccating conditions a reconstructed skin model, said model being obtained from a skin sample from a child; b) bring an active ingredient or candidate formulation into contact with the skin model from step a); c) measure the level of expression of at least one biological marker in the skin model from step b); and d) determine whether said active ingredient or candidate formulation is a formulation or active ingredient for reducing the effects of dehydration of children's skin according to the level of step c).

[0050] Those skilled in the art will readily understand that steps a) and b) can be performed simultaneously or sequentially, as required. In other words, the reconstructed skin model can be cultured under desiccated conditions in step b) in the presence of the active ingredient or formulation. Alternatively, the skin model can first be cultured under desiccated conditions and then exposed to the active ingredient or formulation. By way of illustration, desiccated conditions are cell culture conditions in which the humidity level in the atmosphere is lower than that of typical skin cell culture conditions. For example, the humidity level in the atmosphere is 50% or less. For example, it is 45%, 40%, 35%, 30%, or 25% or less. In one particular example, it is 25% or less.

[0051] In another example, the expression level of said biological marker from step c) is compared to a reference expression level.

[0052] The candidate formulation is a formulation for preventing or reducing the effects of dehydration in children's skin, provided that said candidate formulation modulates 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. For example, it may be advantageous to isolate formulations that minimize the effects of dehydration on markers preferentially expressed in stem cells, as these formulations help preserve the renewal capacity of children's fragile epidermis. Similarly, it would be beneficial to identify formulations that minimize the effects of dehydration on barrier markers in children, in order to maintain the integrity of the skin barrier.Finally, it might be desirable to isolate formulations that do not induce markers of inflammation. For example, a candidate formulation is one for the prevention or reduction of the effects of dehydration in children's skin if said candidate formulation increases or maintains the expression levels of at least one biological marker of barrier function, and / or at least one biological marker preferentially expressed in stem cells, and / or at least one lipid, and / or at least one NMF. Alternatively, or in combination, a candidate formulation is one for the prevention or reduction of the effects of dehydration in children's skin if said candidate formulation decreases or maintains the expression levels of at least one biological marker of inflammation.

[0053] Similarly, the candidate active ingredient is an active ingredient for preventing or reducing the effects of dehydration on children's skin, provided that said candidate active ingredient modulates the expression of at least one biological marker. 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. In a preferred embodiment, the candidate active ingredient is an active ingredient for preventing or reducing the effects of dehydration on children's skin, provided that said candidate active ingredient increases or maintains the expression levels of at least one biological marker of barrier function, and / or at least one biological marker preferentially expressed in stem cells, and / or at least one lipid, and / or at least one NMF.Alternatively or in combination, the candidate active ingredient is an active ingredient for the prevention or reduction of the effects of dehydration of children's skin, if said candidate active ingredient allows for the reduction or maintenance of the expression levels of at least one biological marker of inflammation.

[0054] Initially, the formulation or active ingredient of interest is brought into contact with a reconstructed skin culture obtained from a sample taken from a child. This contact of the active ingredient with the skin model can be performed directly. Alternatively, it may be advantageous to formulate the active ingredient, for example, to obtain a liquid composition, in order to facilitate its contact with the skin model. Thus, by way of illustration, the process further includes a step of formulating the active ingredient, particularly as a liquid solution, especially an aqueous one, prior to the step of bringing said active ingredient into contact with a skin model.

[0055] The inventors have previously shown that the expression profiles of specific gene categories (e.g., barrier, inflammation, defense, and stem cell genes) change with age (WO application 2014 / 009566). A person skilled in the art can thus easily characterize the skin at the molecular level from birth to adulthood. In particular, a person skilled in the art will note that children's skin cells exhibit a specific expression profile of genes involved in particular physiological processes, including cellular metabolism, stress response, inflammation, immunity, apoptosis, growth / proliferation and the cell cycle, cell signaling, migration and differentiation, the epidermal barrier, adhesion, and skin pluripotent stem cells.

[0056] The reconstructed skin model obtained from a skin sample from a child can be any tissue model comprising skin cells, including keratinocytes, and in which said skin cells were obtained from a sample from a child.

[0057] The term "skin sample" here refers to any sample containing skin cells. Skin samples therefore include fresh skin explants obtained directly from the patient, as well as skin cell cultures in suspension, monolayer skin cell cultures, bilayer skin cell cultures, and tissue models, including reconstructed skin cultures and reconstructed mucous membrane cultures. Since working with fresh explants is often difficult, using skin cell cultures is particularly advantageous. Skin cells can include normal, healthy, or pathological cells, or cells derived from cell lines. For example, skin cells used in culture may be cells obtained from skin tissue explants.By "explant" or "skin explant" we mean here a removal of cells or skin tissue, which may be carried out for surgical purposes or to perform analyses.

[0058] In particular, an explant can be obtained during a surgical excision. "Excision" here refers to a surgical procedure involving the removal (cutting out) of a more or less wide or deep section of skin to treat an abnormality or growth. Excision is performed either to remove a cancerous or suspected cancerous tumor, or to treat a benign skin abnormality that is bothersome, whether for functional or aesthetic reasons. Examples of excision include skin samples obtained after plastic surgery (breast augmentation, abdominoplasty, facelift, preputial removal, otoplasty, i.e., ear pinning, syndactyly or supernumerary finger removal, etc.).

[0059] An explant can also be obtained by biopsy. Here, "biopsy" refers to the removal of cells or skin tissue for analysis. Several types of biopsy procedures are known and practiced. The most common types include (1) incisional biopsy, in which only a tissue sample is taken; (2) excisional biopsy (or surgical biopsy), which involves the complete removal of a tumor mass, thus performing both a therapeutic and diagnostic procedure; and (3) needle biopsy, in which a tissue sample is taken with a needle, which may be either large or small. Other types of biopsy exist, such as smears or curettage, and are also included here.

[0060] Alternatively, these skin cells can be obtained by stem cell differentiation (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).

[0061] Skin cells, whether derived from a biopsy or obtained by stem cell differentiation, comprise at least one cell type normally present in the hypodermis, dermis, and / or epidermis. These cells include, among others, keratinocytes, melanocytes, fibroblasts, adipocytes, endothelial cells, mast cells, Langerhans cells, and / or Merkel cells. Preferably, the skin cells according to the invention comprise at least keratinocytes or fibroblasts. By way of illustration, the skin cells comprise keratinocytes and / or fibroblasts.

[0062] Many methods for culturing skin cells are known to those skilled in the art. Any of these methods can be used to culture the skin cells of the invention. Advantageously, the skin cells are cultured and / or preserved under conditions that maintain, at least partially, cellular metabolism and / or cellular functions. The skin cell culture described herein therefore includes both suspension skin cell cultures, monolayer skin cell cultures, and bilayer skin cell cultures, as well as tissue models, including reconstructed skin cultures and reconstructed mucous membrane cultures.

[0063] For example, skin cell cultures in suspension have been routinely performed in a large number of laboratories for several decades. Similarly, monolayer and bilayer skin cell cultures have been known and used for a very long time.

[0064] Furthermore, numerous tissue models, including in particular reconstructed skin models and reconstructed mucous membrane models (Rosdy et al., In Vitro Toxicol., 10(1): 39-47, 1997; Ponec et al., J Invest Dermatol., 109(3): 348-355, 1997; Ponec et al., Int J Pharm., 203(1-2): 211-225, 2000; Schmalz et al., Eur J Oral Sci., 108(5): 442-448, 2000; Black et al., Tissue Eng, 11(5-6): 723-733, 2005; Dongari-Batgtzoglou and Kashleva, Nat Protoc, 1(4): 2012-2018, 2006; Bechtoille et al., Tissue Eng, 13(11): 2667-2679, 2007; Vrana et al., Invest Ophthalmol Vis Sci, 49(12): 5325-5331, 2008; Kinicoglu et al., Biomaterials, 30(32): 6418-6425, 2009; Auxenfans et al., Eur J Dermatol, 19(2): 107-113, 2009; Kinicoglu et al., Biomaterials, 32(25): 5756-5764, 2011; Costin et al., 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 B1; EP 1 878 790 B1; EP 1 974 718; US 2007 / 0148,771; US ​​2010 / 0,099,576; WO 02 / 070729; WO 2006 / 063864; WO 2006 / 0,63865; WO 2007 / 064305) are available to those skilled in the art.

[0065] Advantageously, the tissue model includes reconstructed skin models and reconstructed mucosal models. For example, the reconstructed skin model is selected from the group comprising dermal models, containing mainly stromal cells, and more specifically fibroblasts; epidermal models, consisting mainly of keratinocytes; hypodermal models; skin models comprising a dermis and an epidermis; and skin models comprising a dermis, an epidermis, and a hypodermis. Models containing at least one dermis form connective-type tissues, while models containing at least one epidermis form stratified epithelia comprising the characteristic layers of the tissue in question. For example, a basal layer ( basal layer ), a thorny layer ( thorny bed ), a granular layer ( granular layer ), and a stratum corneum ( stratum corneum ). On the other hand, the reconstructed mucosal model is, for example, a model of the mucosa of the mouth, gum, vagina or cornea.

[0066] Advantageously, said model is a connective tissue model of dermal matrix comprising a matrix support preferably selected from: an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon membrane or sponge, a semi-permeable polycarbonate or polyethylene, polypropylene, polyethylene terephthalate (PET) membrane, a semi-permeable Anopore inorganic membrane, cellulose acetate or ester (HATF) membrane, a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane, a polyglycolic acid membrane or film.

[0067] This group includes, for example, the Skin 2™< model ZK1100 and Dermagraft ®< and Transcyte ®< (Advanced Tissue Sciences); a cell culture treated plastic (formation of a dermal sheet: Michel et al., In Vitro Cell. Dev Biol. -Animal, 35: 318-326, 1999); a gel or membrane based on hyaluronic acid (Hyalograft ®< 3D - Fidia Advanced Biopolymers) and / or collagen (such as a dermis equivalent or collagen lattice) and / or fibronectin and / or fibrin; in this group we find for example the Vitrix ®< dermal model (Organogenesis); a porous matrix, surfaced or unsurfaced (for example a dermis equivalent), made from collagen which may contain one or more glycosaminoglycans and / or possibly chitosan (EP0296078A1, WO 01 / 911821 and WO 01 / 92322).

[0068] This group includes, for example, the dermal model Mimederm ®< (Coletica).

[0069] These matrix structures include stromal cells, particularly fibroblasts.

[0070] Advantageously, said skin model is an epidermal model comprising a matrix support preferably chosen from: an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon membrane or sponge, a semi-permeable polycarbonate or polyethylene membrane, polypropylene, polyethylene terephthalate (PET), a semi-permeable Anopore inorganic membrane, cellulose acetate or ester (HATF), a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane; This group includes 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.

[0071] In this group, we can particularly mention the models: Laserskin ®< (Fidia Advanced Biopolymers), Episkin ®< (L'Oréal).

[0072] These models can be seeded by fibroblasts in the dermal part.

[0073] These models, which may or may not include fibroblasts, serve as a scaffold 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.

[0074] Advantageously, said tissue model is a reconstructed skin or mucous membrane tissue model comprising a dermal or chorionic matrix support preferably selected from:an inert support selected from the group consisting of a semi-permeable synthetic membrane, in particular a semi-permeable nitrocellulose membrane, a semi-permeable nylon membrane, a Teflon membrane or sponge, a semi-permeable polycarbonate or polyethylene, polypropylene, or polyethylene terephthalate (PET) membrane, a semi-permeable Anopore inorganic membrane, a cellulose acetate or ester (HATF) membrane, a semi-permeable Biopore-CM membrane, a semi-permeable polyester membrane, said inert support containing or not containing stromal cells, in particular fibroblasts, a collagen and / or hyaluronic acid and / or fibronectin-based gel, and / or fibrin-based gel comprising stromal cells, in particular fibroblasts, a porous matrix, surfaced or unsurfaced, made from collagen which may contain one or more glycosaminoglycans and / or optionally chitosan, these porous matrices incorporating stromal cells,in particular fibroblasts, a de-epidermised or dead dermis, human or animal.

[0075] In this group, we can particularly cite the models Mimeskin (Coletica), EpidermFT ™<, EpiAirway ™<, EpiOccular ™< EpiOral ™<, EpiGingival ™<, EpiVaginal ™< (MatTek corporation), Human Corneal Epithelium (HCE), Human Oral Epithelium (HOE), Human Gingival Epithelium (HGE), Human Vaginal Epithelium (HVE) (Skinethic ®< ), Phenion ®< Full Thickness Skin Model (Phenion) Apligraf ®< (Organogenesis), ATS-2000 (CellSystems ®< Biotechnologie Vertrieb) as well as Skin 2TM (ZK1200-1300-2000 Advanced Tissue Science).

[0076] There are also models dedicated to tissue therapy that can also be used within the framework of the present invention. Examples include the Epidex (Modex Thérapeutiques), Epibase ®< (Laboratoire Genévrier), Epicell ™< (Genzyme), Autoderm ™< and Transderm ™< (Innogenetics) models.

[0077] The matrix support is then seeded by keratinocytes to rebuild the epidermis and ultimately obtain reconstructed skin.

[0078] Advantageously, the skin model used includes a model in which at least one complementary cell type has been incorporated, such as endothelial cells (ECs) and / or immune cells such as lymphocytes, macrophages, mast cells, dendritic cells and / or adipose cells and / or skin appendages, such as hair, scalp hair, sebaceous glands.

[0079] After exposing the reconstructed skin model to desiccation conditions, a person skilled in the art can proceed to measure the level of expression of biological markers.

[0080] The term "biological marker" here refers to a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biological marker thus encompasses a wide range of substances and diverse parameters. For example, a biological marker can be a substance whose detection indicates a particular pathological state (e.g., the presence of activated protein C as a marker of infection), or conversely, a substance whose detection indicates a specific physiological state. Examples of biological markers include a gene, gene products such as its transcripts and peptides derived from its transcripts, a lipid, a sugar, or a metabolite.

[0081] For illustrative purposes, a biological marker is a gene, the products of a gene such as transcripts or peptides, a lipid, a sugar or a metabolite whose changes in expression, particularly level of expression, correlate with a physiological state of the child's skin.

[0082] A person skilled in the art seeking to determine which class a gene or protein marker belongs to can easily consult the relevant scientific literature or refer to public databases such as, for example, those grouped on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0083] The inventors specifically selected markers whose expression levels change surprisingly and unexpectedly in children after dehydration. These selected markers are therefore of particular interest within the framework of the invention's method, since their expression levels are measured on a skin model reproducing the characteristics of children's skin.

[0084] The inventors demonstrated, in particular, that desiccation conditions affected the amount of lipids and NMF produced by a child's skin model. Furthermore, they showed that, simultaneously, the expression of barrier markers decreases, as does that of NMF synthesis markers. Finally, stem cell markers are also affected, and consequently, so is the epidermal renewal capacity. Conversely, contacting the skin with a formulation active against the effects of skin dehydration prevents and corrects variations in the expression of these markers, thus highlighting their relevance.

[0085] The biological marker is therefore advantageously a marker selected from the group of lipids of the stratum corneum, including ceramides, NMFs, markers of inflammation, markers of barrier function and markers preferentially expressed in stem cells.

[0086] The inventors specifically demonstrated that the amount of skin lipids decreased when a child's skin model was cultured under desiccated conditions. In the stratum corneum, lipids are arranged in lamellar layers within the spaces between corneocytes, forming a cement that helps protect the skin against external aggressions and maintain a good level of intraepidermal water. These lipids are phospholipids, cholesterol, and glucosylceramides, which are modified in the intercorneocyte spaces by specialized enzymes into ceramides, cholesterol, cholesterol sulfate, and free fatty acids (Jungerstend et al., Contact Dermitis, 58(5): 255-262, 2008). Ceramides, sterols, and free fatty acids are also found in sebum, among other components.Finally, the skin's surface is covered by a protective film that allows the skin to maintain its hydration and protect itself against external aggressors. This surface skin film includes, among other things, a hydrolipidic film, which is composed primarily of sweat, water, sebum, and other lipids. These lipids include ceramides, triglycerides, and fatty acids in roughly equal proportions.

[0087] In a specific example, the biological marker is therefore a lipid marker. By "lipids," we mean any naturally occurring fat-soluble (i.e., lipophilic) molecule. Lipids are a heterogeneous group of compounds with many essential biological functions. Lipids can be further defined as small hydrophobic or amphiphilic molecules that originate entirely or partially from ketoacyl or isoprene groups. For an overview of all lipid classes, see the "Lipid Metabolites and Pathways Strategy (LIPID MAPS) classification system" (National Institute of General Medical Sciences, Bethesda, MD). In particular, the lipid marker is selected from ceramides, phospholipids, glucosylceramides, sterols, triglycerides, and free fatty acids. By way of illustration, the lipid marker of the invention is selected from ceramides.

[0088] A "ceramide" is a lipid resulting from the amidation reaction of sphingosine and a fatty acid. A ceramide therefore consists of a sphingosine or phytoshingosine base linked by an amide bond to α-hydroxy, ω-hydroxy, or non-hydroxy acids possessing varying hydrophobic chain lengths. In the stratum corneum In humans, nine classes of ceramides, designated CER 1 to 9, have been identified (see, for example, Dayan, Cosm & Toil, 121(1): 37-44, 2006; Jungerstend et al., Contact Dermitis, 58(5): 255-262, 2008; Farwick et al., Cosm & Toil, 124(2): 63-72; Masukawa et al., J Lipid Res., 50(8): 1708-1719, 2009). The ceramide is, for example, selected from the group consisting of these CER1 to 9 ceramides.

[0089] Furthermore, the inventors have shown that NMFs are affected by dehydrating conditions. Natural Moisturizing Factor (NMF) is produced by the proteolysis of filaggrin through a cascade of reactions involving enzymes, including caspase 14 and peptidylarginine deiminase (PAD1). NMF is a mixture of hygroscopic substances with the property of retaining water (Fluhr et al., Exp Dermatol., 19(6): 483-492, 2010). Among these, the sodium salt of pyrrolidone carboxylic acid, or PCA Na (derived from the cyclization of glutamic acid released by the decomposition of filaggrin), and lactates are the most hygroscopic substances. NMF also includes free amino acids (serine, citrulline...), citrates and formates, urea, ions, nitrogen, uric acid, glycosamine, creatinine, phosphates, as well as compounds not yet identified.The amount of NMF can be measured by all methods known to those skilled in the art, including Raman microspectroscopy. For illustrative purposes, the biological marker is therefore NMF.

[0090] The inventors have thus shown that markers of inflammation are particularly expressed after children's skin has been incubated in a dry atmosphere. Inflammation is a normal defense reaction of the body, but it can contribute to a decrease in skin integrity. Furthermore, the inventors have shown that, at the same time, dehydration induces a reduction in the expression of barrier function markers and markers preferentially expressed in stem cells in a children's skin model.

[0091] By "markers of skin inflammation", we mean here markers whose variation in expression correlates with skin inflammation.

[0092] Inflammation refers to all the body's defense mechanisms by which it recognizes, destroys, and eliminates foreign substances. Cutaneous inflammation, more specifically, is an immune system reaction to an injury to the skin, such as an environmental irritant, whether or not it results in a wound, or even vascular damage. Cutaneous inflammation can manifest as erythema, characterized by redness associated with local vasodilation, edema, characterized by swelling, and a sensation of heat. Furthermore, cutaneous inflammation is accompanied by a change in the expression level or concentration of gene or protein markers well known to those skilled in the art, who can refer, for example, to Vahlquist (Acta Derm Venereol; 80: 161; 2000).

[0093] The initiation and continuation of inflammation, and its spread from the initial site, involve factors that are either synthesized locally or exist as inactive precursors in the circulation. Depending on the type of mediators synthesized, specific processes can be distinguished within the inflammatory response. Thus, cutaneous inflammation encompasses at least three distinct processes: protein inflammation, lipid inflammation, and neurogenic inflammation.

[0094] By "protein inflammation" or "nonspecific inflammation," we mean the production, in response to an external insult, of protein-based inflammatory mediators, such as the cytokines IL-1, IL-2, IL-6, IL-8, and TNFα, the complement system, or proteins involved in coagulation, as appropriate. By "lipid inflammation," we mean the production, in response to said external insult, of lipid-based mediators, notably prostaglandins and leukotrienes, both synthesized from arachidonic acid, as well as the activation of the enzymes responsible for this production (Shimizu, Annu Rev Pharmacol Toxicol., 49: 123-150, 2009). The protein and lipid mediators thus produced induce a cascade of reactions within the skin involving other inflammatory cells, particularly immune and vascular cells. The clinical result is manifested, in particular, by redness and even edema.

[0095] In response to external aggression, the neurosensory system can be stimulated and associated with an inflammatory reaction involving other cellular components such as nerves (or nerve endings) and cells like mast cells. "Neurogenic inflammation" refers to the release by nerve endings, in response to external aggression, of specific mediators, including neuropeptides (notably tachykinins such as substance P and Calcitonin Gene-Related Peptide or CGRP). The activity of specific receptors, such as the substance P receptor or the TRPV1 receptor, also contributes to neurogenic inflammation according to the invention. Neurogenic inflammation most often results in a sensation of pain and / or discomfort and / or itching (pruritus).

[0096] The marker of skin inflammation is, for example, chosen from among markers of protein inflammation, markers of lipid inflammation and markers of neurogenic inflammation.

[0097] For illustrative purposes, the protein marker for inflammation is chosen from the group consisting of interleukins, preferably IL-1α and IL-8. Human interleukin IL-1α has a protein sequence represented by the NCBI reference sequence: NP_000566. This protein is encoded by the human IL-1A gene (NCBI reference: Gene ID: 3552), whose sequence corresponds to the NCBI reference: NM_000575. The protein sequence of human interleukin IL-8 corresponds to the NCBI reference sequence: NP_000575. This protein is encoded by the human IL-8 gene (NCBI reference: Gene ID: 3576). Its sequence is available under the NCBI reference: NM_000584.

[0098] The nerve growth factor receptor NGFR has a protein sequence represented by the reference sequence NP_002498. This protein is encoded by the NGFR gene (NCBI reference: Gene ID: 4804). Its sequence is available under NCBI reference: NM_002507.

[0099] The OSMR receptor (Oncostatin-M specific receptor subunit beta or oncostatin M receptor) is a type I cytokine receptor whose ligand is oncostatin. The OSMR protein corresponds to the sequence represented by Genbank accession number NP_001161827 and is encoded by the OSMR gene (NCBI reference: Gene ID: 9180), with sequence NM_001168355.

[0100] The F2RL1 protein (coagulation factor II (thrombin) receptor-like 1), also known as PAR2 (protease-activated receptor 2) or GPR11 (G-protein coupled receptor 11), is a receptor encoded by the F2RL1 gene (NCBI reference: Gene ID: 2150) in humans. This receptor, which belongs to the GPCR family of 7-transmembrane domain receptors, plays a role in modulating inflammatory responses. The sequence of this protein is available under NCBI reference: NP_005233, while the F2RL1 gene sequence can be obtained using GenBank accession number NM_005242.

[0101] The marker of lipid inflammation is advantageously chosen from among prostaglandins, in particular prostaglandin E2, and the enzymes of their synthesis from arachidonic acid, in particular PTGS2.

[0102] Prostaglandin E2 (PGE2) is a well-known derivative of arachidonic acid produced by the action of cyclooxygenase. There are two isoforms of cyclooxygenase (COX): cyclooxygenase 1, which is constitutive in tissues, and cyclooxygenase 2, which is induced by inflammatory processes. Pro-inflammatory stimulation (trauma, cytokines, etc.) leads to the synthesis of PGE2, which is responsible for vasodilation (generating redness and edema), sensitization of nociceptors to bradykinin and histamine (responsible for pain), and fever (in conjunction with the cytokines IL-1 and IL-6).

[0103] The enzyme cyclooxygenase 2, also known as prostaglandin-endoperoxide synthase (PTGS), is encoded by the human PTGS2 gene (NCBI reference: Gene ID: 5743). The sequence of this gene is available under NCBI reference: NM_000963 and the protein sequence under NCBI reference: NP_000954.

[0104] As an illustration, the marker of neurogenic inflammation is chosen from among neuropeptides and neuropeptide receptors, specifically the TRPV1 and SRP receptors. The TRPV1 (Transient Receptor Potential Vanilloid 1) receptor is a cation channel membrane protein of the TRP family with NCBI reference sequence NP_061197. In the skin, TRPV1 is expressed by keratinocytes, mast cells, and nerve fibers. In response to an irritant, TRPV1 activation leads to the production of cytokines and neuropeptides and is a key player in neurogenic inflammation. The gene encoding the TRPV1 receptor is the TRPV1 gene (NCBI reference: Gene ID: 7442), whose sequence has NCBI reference NM_018727. The TRPV3 gene (NCBI reference: Gene ID: 162514), whose sequence has the NCBI reference: NM_001258205, encodes a receptor of the same family as TRPV1. The sequence of this TRPV3 receptor corresponds to the reference sequence NP_001245134.

[0105] Protachykinin-1 is a protein encoded in humans by the TAC1 gene (NCBI reference: Gene ID: 6863), whose sequence is NCBI reference: NM_003182. The sequence of this protein is available under NCBI reference: NP_003173. The substance P receptor (SPR; also known as neurokinin 1 receptor, NK1R, or tachykinin receptor 1, TACR1) is a G protein-coupled receptor (GPCR) that transmits the signaling pathway for substance P (SP) and other tachykinins. It is encoded by the human TACR1 gene, whose sequence is NCBI reference: NM_001058. Its peptide sequence is NCBI reference sequence: NP_001049.

[0106] The MRGPRD receptor is a receptor belonging to the family of 7-transmembrane domain receptors, also known as GPCRs. The MRGD (Mas-related G-protein coupled receptor member D) receptor is specifically expressed in a subpopulation of sensory neurons involved in pain detection. This receptor is encoded by the human MRGPRD gene (NCBI reference: Gene ID: 116512). The gene sequence is available under NCBI reference: NM_198923 and the protein sequence under NCBI reference: NP_944605.

[0107] Phospholipase 2A enzymes represent a family of enzymes that release pro-inflammatory mediators (lysophospholipids, oxidized fatty acids) by hydrolyzing short-chain oxidized phospholipids present on oxidized low-density lipoproteins (LDL). Among these, Phospholipase A2 Group II F is encoded by the human PLA2G2F gene (NCBI reference: Gene ID: 64600). The gene sequence is available under NCBI reference: NM_022819 and the protein sequence under NCBI reference: NP_073730.3

[0108] As an example, the marker of skin inflammation is therefore chosen from the group consisting of IL1A, IL8, PTGS2, NGFR, TAC1, TAC1R, TRPV1, TRPV3, MRGPRD, OSMR, PLA2G2F and F2RL1.

[0109] The present inventors have also shown that dehydration induces a reduction in the expression of barrier function markers and markers preferentially expressed in stem cells.

[0110] "Barrier markers" include markers that are specifically expressed in the outermost layers of the epidermis and that participate in the barrier function.

[0111] As is well known to those skilled in the art, the skin's primary function is to establish a protective barrier against environmental damage while allowing some exchange between the internal and external environments. This barrier function is mainly carried out by the stratum corneum of the epidermis. Lipids and intercellular corneodesmosomes as well as the corneal envelope of corneocytes are its key constituents.

[0112] However, under the stratum corneum, Tight joints constitute a second barrier function. These joints form at the level of the granular layerA selective paracellular diffusion barrier prevents the penetration of harmful molecules. Tight junctions are composed of various transmembrane proteins, including claudins, occludin, and ZO1.

[0113] The barrier functions provided by the stratum corneum and tight junctions are closely linked. Indeed, the alteration of one or the other can influence the formation of the other.

[0114] For example, barrier function markers are markers expressed in the stratum corneum or markers expressed in the tight junctions of the granular layer. For example, said epidermal barrier marker is selected from the group consisting of desmoglein 1 (DSG), sciellin (SCEL), peptidyl arginine deiminase 1 (PADI1), caspase 14 (CASP14), loricrin (LOR), transglutaminase 1 (TGM1) and claudin 1 (CLDN1).

[0115] The corneodesmosome is the only junctional structure of the stratum corneum, highlighting its importance in maintaining the integrity of the stratum corneum. Desmoglein-1 is a constituent protein of corneodesmoses, and its sequence is available under the reference NP_001933. The DSG1 gene (NCBI reference: Gene ID: 1828) encodes desmoglein-1 and has the reference sequence NM_001942. Sciellin, with the sequence NP_001154178, and encoded by the SC gene (NCBI reference: Gene ID: 8796), which itself has the sequence NM_001160706, is a precursor of the corneal envelope. Loricrin is a major protein component of the corneal envelope, representing approximately 70% of its mass. This protein has the sequence NP_000418 and is encoded by the LOR gene (NCBI reference: Gene ID: 4014) with sequence NM_000427. The formation of bonds between the protein components of the corneal envelope is ensured by the enzyme transglutaminase 1.The sequence of this enzyme corresponds to that which can be found under the reference NP_000350. The TGM1 gene (reference NCBI: Gene ID: 7051) codes for desmoglein-1 and has the reference sequence NM_000359.

[0116] Tight junctions represent one of the modes of cell adhesion in epithelial tissues. They block the flow of fluid between cells, thus ensuring a seal between two tissue compartments. They are located at the apex of epithelial cells, where they form a continuous band around the entire surface, providing this seal. The CLDN1 gene (NCBI reference: Gene ID: 9076) encodes the claudin-1 protein, which is one of the most important components of tight junctions. This protein has a sequence corresponding to that with NCBI reference NP_066924. The CLDN1 gene sequence is available under reference NM_021101.

[0117] Barrier markers also include markers of NMF and intercorneocyte lipid metabolism. These compounds help retain water in the epidermis as it ascends to the stratum corneum. Peptidylarginine deiminase 1 (PADI1) and caspase 14 (CASP14) are two enzymes involved in the maturation of filaggrin, which leads to the formation of NMF. Peptidylarginine deiminase 1, with sequence NP_037490, is an enzyme that removes an imine group from filaggrin and K1 keratin, which maintains the hydration of the stratum corneum and, consequently, the skin barrier function. This enzyme is encoded by the PADI1 gene, whose sequence (NCBI reference: Gene ID: 29943) can be found under the reference NM_013358. Caspase-14 is a member of the caspase family that is required for the degradation of filaggrin into NMF (Hoste et al., J Invest Dermatol. 131(11): 2233-2241, 2011).This protein, with sequence NP_036246, is encoded by the CASP14 gene (NCBI reference: Gene ID: 23581) whose sequence is found under reference NM_012114.

[0118] The term “preferentially expressed stem cell markers” encompasses markers, and more specifically genes and proteins, that are specifically present in epidermal stem cells.

[0119] The term "epidermal stem cell" or "epidermal stem cell" refers to an epidermal cell capable of long-term renewal. Epidermal stem cells include, among others, follicular stem cells, sebaceous stem cells, and basal stem cells, the latter also being called interfollicular epidermal stem cells. "Follicular stem cells," "sebaceous stem cells," and "basal stem cells" refer to stem cells located, respectively, in the bulge region of the hair follicle, in the sebaceous glands, and in the basal layer of the epidermis. For example, the epidermal stem cells of the invention are basal stem cells.

[0120] More precisely, an epidermal stem cell is defined as a cell with a high long-term renewal potential. "Renewal potential" here refers to the capacity to undergo at least one cell division cycle. A "high long-term renewal potential" therefore represents a cell's ability to enter several successive cell division cycles. It is well known that differentiated skin cells are not capable of performing multiple successive divisions (Fortunel and Martin, J Soc Biol, 202(1): 55-65, 2008). It is understood here that "successive" does not mean "consecutive," and that there can be periods during which a stem cell remains quiescent without losing its high long-term renewal potential.

[0121] The preservation of a high long-term renewal potential is expressed through asymmetric division, producing two different cell types. The first daughter cell is a stem cell identical to the parent stem cell, while the second is a transient amplifying cell that divides in a limited way over a short period before entering the differentiation process. Advantageously, epidermal stem cells are therefore also capable of generating at least one type of epidermal cell through differentiation. In other words, the transient amplifying cell is capable of giving rise to at least one type of epidermal cell through differentiation. For example, such an epidermal cell is a keratinocyte. As an illustration, the transient amplifying cell is capable of giving rise to all types of epidermal cells through differentiation.

[0122] As an illustration, the markers expressed in stem cells are markers that participate in the functions and protection of stem cells. Examples include the markers ΔNp63, BIRC5 (survivin), FN1 (fibronectin 1), MCSP (Melanoma-associated Chondroitin Sulfate Proteoglycan), LRIG1 (Leucine-rich repeats and immunoglobulin-like domains protein 1), GJA1 (connexin 43), NID1 (nidogen 1), KRT15 (keratin 15), KRT19 (keratin 19), EGFR (epidermal growth factor receptor), CD71 (transferrin receptor), DSG3 (desmoglein 3), ITGB1BP1 (integrin beta1 binding protein), ITGA6 (integrin alpha 6), ITGB1 (integrin beta1), and ITGB4 (integrin beta 4), as well as markers involved in signaling and stem cell activity regulation such as Wnt / beta catenin, sonic hedgehog (SHH), and NOTCH1 (Notch homolog). 1, translocation-associated).The markers ΔNp63 and survivin are markers of resistance to apoptosis, thus playing a role in stem cell survival. Cytokeratins 15 and 19 are positive markers of stem cells, with cytokeratin 15 being a survival marker. MCSP colocalizes with integrins in non-dividing cells, while integrin beta1 (a marker of adhesion to the extracellular matrix of the basement membrane) and integrin alpha6 (a constituent of hemidesmosomes and a marker of keratinocyte binding to one another) are surface proteins that participate in intercellular communication, regulate differentiation / proliferation processes, and interact with the cell niche. The transferrin receptor CD71 is a known stem cell surface marker, used to isolate highly clonogenic cells within a population of integrin-alpha6-positive cells.Finally, Lrig1 is an antagonist of the Epidermal Growth Factor (EGFR) receptor, thus keeping stem cells quiescent, whereas the EGFR receptor, which is a marker whose absence characterizes stem cells, on the contrary leads cells into the proliferation pathway.

[0123] For illustrative purposes, the marker preferentially expressed in stem cells is chosen from the group consisting of the markers ΔNp63, KRT15 (keratin 15), KRT19 (keratin 19), BIRC5 (survivin), and NOTCH1 (Notch Homolog 1). These markers are well known to those skilled in the art. The genes KRT15 (NCBI reference: Gene ID: 3866), KRT19 (NCBI reference: Gene ID: 3880), BIRC5 (NCBI reference: Gene ID: 332), and NOTCH1 (NCBI reference: Gene ID: 4851) correspond to the sequences represented by the following GenBank accession numbers: NM_002275, NM_002276, NM_001012270, and NM_017617, respectively. The proteins keratin 15, keratin 19, survivin and Notch homolog 1 correspond to the sequences represented by the following Genbank accession numbers: NP_002266, NP_002267, NP_001012270 and NP_060087, respectively.The ΔNp63 protein is an isoform of the TP63 (Tumor protein 63) protein obtained by transcription from an internal promoter located in an intron. This protein corresponds to the sequence represented by the Genbank accession number: NP_001108452, and is encoded by the TP63 gene (NCBI reference: Gene ID: 8626), the sequence of which is available under the following Genbank accession number: NM_001114980.

[0124] It will also be evident to a person skilled in the art that the method of the invention will allow an evaluation of the effectiveness of the formulation or active ingredient that is all the more complete as a large number of markers of different types are used.

[0125] The method described herein may include a step (c) of measuring the expression level of a combination of biological markers. For example, this combination may include at least two markers, these markers being chosen from at least two different categories of markers described above: NMF, lipids (including ceramides), inflammation markers, barrier markers, and stem cell markers. Alternatively, this combination may include more than two markers. For illustrative purposes, each marker belongs to a distinct category of markers described above. It is also possible to use marker combinations as defined above, in which certain classes of markers are represented by more than one marker.

[0126] The use of marker combinations including at least one marker of each of the different types indicated above is particularly advantageous.

[0127] For each of these markers, the term "expression level" refers to the cellular concentration of that marker. Thus, the expression level of ceramides corresponds to the concentration of those lipids in the cell. If the marker is a gene, the "expression level" corresponds to the cellular concentration of at least one of the gene products of that marker. More precisely, the expression level of that biological marker corresponds to the quantity or cellular concentration of the transcript of that gene or of the protein produced by that transcript. For example, the expression level of that biological marker corresponds to the quantity or cellular concentration of the transcript of that gene. As another example, the expression level of that biological marker corresponds to the quantity or cellular concentration of the protein produced by that transcript.

[0128] The term "measuring the expression level of a combination of biological markers" refers to measuring the expression level of each marker in the combination. Gene expression can be measured, for example, at the nucleotide level by measuring the quantity of transcripts of that gene, and can also be measured, for example, at the peptide level by measuring the quantity of proteins derived from those transcripts. Thus, "measuring the expression level of that gene" means measuring the quantity of the gene product in its peptide or nucleotide form.

[0129] In general, the expression of the biological marker will be detected in vitro based on the reconstructed skin model.

[0130] The procedure described herein may include one or more intermediate steps between obtaining the reconstructed skin model and measuring the expression of the biological marker. These steps correspond to the extraction from the reconstructed skin model of a lipid sample, an NMF sample, an mRNA (or corresponding cDNA) sample, or a protein sample. This sample can then be used directly to measure marker expression. The preparation or extraction of mRNA (and its reverse transcription into cDNA), proteins, lipids, or NMF from a skin cell sample are routine procedures well known to those skilled in the art.

[0131] Once a sample of mRNA (or corresponding cDNA) or protein is obtained, the expression of the marker can be measured either in the mRNAs (i.e., in all the mRNAs or cDNAs present in the sample) or in the proteins (i.e., in all the proteins present in the sample). The method used for this depends on the type of transformation (mRNA, cDNA, or protein) and the type of sample available.

[0132] When marker expression is measured at the level of the corresponding mRNA (or cDNA), any technology commonly used by a person skilled in the art can be implemented. These gene expression level analysis technologies, such as transcriptome analysis, include well-known methods such as PCR (Polymerase Chain Reaction, if starting from DNA), RT-PCR (Reverse Transcription-PCR, if starting from RNA), or quantitative RT-PCR, or even nucleic acid microarrays (including DNA microarrays and oligonucleotide microarrays) for higher throughput.

[0133] The term "nucleic acid chips" refers to several different nucleic acid probes attached to a substrate, which may be a microchip, a glass slide, or a microsphere-sized bead. The microchip may be made of polymers, plastics, resins, polysaccharides, silica or a silica-based material, carbon, metals, inorganic glass, or nitrocellulose.

[0134] The probes can be nucleic acids such as cDNA (“cDNA chip”), mRNA (“mRNA chip”) or oligonucleotides (“oligonucleotide chip”), said oligonucleotides typically being between approximately 25 and 60 nucleotides in length.

[0135] To determine the expression profile of a particular gene, a nucleic acid corresponding to all or part of that gene is labeled and then brought into contact with the chip under hybridization conditions. This leads to the formation of complexes between the labeled target nucleic acid and probes attached to the chip surface that are complementary to that nucleic acid. The presence of these labeled hybridized complexes is then detected.

[0136] These technologies make it possible to monitor the expression level of a particular gene, several genes, or even all the genes in the genome (full genome or full transcriptome) in a biological sample (cells, tissues, etc.). These technologies are routinely used by those skilled in the art, and therefore there is no need to describe them in detail here. Examples of implementations of the invention based on gene expression analysis (cDNA microarrays) and quantitative PCR are described in the experimental section.

[0137] Alternatively, any current or future technology capable of determining gene expression based on the amount of mRNA in the sample can be used. For example, a person skilled in the art can measure gene expression by hybridization with a labeled nucleic acid probe, such as Northern blot (for mRNA) or Southern blot (for cDNA), as well as by techniques such as Serial Analysis of Gene Expression (SAGE) and its derivatives, such as LongSAGE, SuperSAGE, DeepSAGE, etc. Tissue microarrays (also known as TMAs) can also be used. Tests commonly used with tissue microarrays include immunohistochemistry and fluorescence in situ hybridization (FISH). For mRNA analysis, tissue microarrays can be coupled with FISH.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"). Several massive parallel sequencing methods are available for this purpose. 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.

[0138] When marker expression is measured at the protein level, it is possible to use specific antibodies, particularly in well-known technologies such as immunoprecipitation, immunohistochemistry, western blot, dot blot, ELISA or ELISPOT, protein chips, antibody chips, or tissue chips coupled with immunohistochemistry.Other techniques that can be used include FRET or BRET techniques, microscopy or histochemistry methods, including confocal microscopy and electron 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), the atomic force microscope, and radio frequency methods, such as multipolar, confocal and non-confocal resonance spectroscopy, fluorescence detection, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., by surface plasmon resonance, ellipsometry, resonant mirror method, etc.).), flow cytometry, radioisotope or magnetic resonance imaging, SDS-PAGE analysis; HPLC-Mass spectrophotometry, liquid chromatography / mass spectrophotometry / mass spectrometry (LC-MS / MS). All these techniques are well known to those skilled in the art and it is not necessary to detail them here.

[0139] If the biological marker is a lipid, particularly a ceramide, a person skilled in the art can use all available methods to measure the lipid content in a sample of skin cells. These methods include, among others, high-performance liquid chromatography (HPLC, see for example Sullivan et al., Arch Ophthalmol., 120(12): 1689-99, 2002), for example coupled with an evaporative light diffraction detector (HPLC-ESD, see Nordbäck et al., J. High Resolut. Chromatogr., 22: 483-486, 1999; Torres et al., J. Chromatogr. A., 1078: 28-34, 2005); thin-layer chromatography (TLC, e.g. Downing et al., J Invest Dermatol., 77(4): 358-360, 1981; Nordstrom et al., J Invest Dermatol., 87(2): 260-263, 1986); nuclear magnetic resonance (NMR, see e.g. Robosky et al., J Lipid Res., 49(3): 686-692, 2008); in vivo confocal Raman microspectroscopy; mass spectrometry, gas chromatography coupled with mass spectrometry (GC-MS, see O'Neill et al., J Chromatogr Sci., 14(1): 28-36, 1976); liquid chromatography coupled with mass spectrometry (see, for example, van Smeden et al., J Lipid Res, 52(6):1211-1221, 2011); Ultra-high-performance liquid chromatography (UPLC, see Rainville et al., J Proteome Res., 6(2):552-558, 2007; Castro-Perez et al., J Proteome Res., 10(9): 4281-4290, 2011). The organization of these lipids in the skin, and more specifically in the . stratum corneum(or stratum corneum), lamellar or lateral organization, by techniques such as X-ray diffraction (Bouwstra et al., J Invest Dermatol., 97(6): 1005-1012, 1991; van Smeden et al., J Lipid Res., 52(6):1211-1221, 1991) or by Fourier transform infrared spectroscopy (Gorcea et al., Int J Pharm. Nov 10, 2011) or by morphometric analysis by electron microscopy (Daehnhardt-Pfeiffer et al., Skin Pharmacol Physiol., 25(3): 155-161, 2012) or by electron microscopy analysis of vitreous skin sections combined with molecular analysis (Iwai et al., J Invest Dermatol., Apr 26, 2012).

[0140] NMF measurement is a well-known procedure for those skilled in the art. In particular, NMF can be measured using in vivo confocal Raman microspectroscopy. This procedure has been commonly used in the field for at least 15 years. Examples include the publications by Caspers et al. (J Invest Dermatol., 116(3): 434-442, 2001), Vyumvuhore et al. (J Biomed Opt., 19(11): 111603, 2014), and Falcone et al. (Skin Pharmacol Physiol, 28: 307-317, 2015). NMF can also be measured by liquid chromatography coupled with mass spectrometry. See, for example, 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 Yang (Application of biophysics and bioengineering to the assessment of skin barrier function.Thesis (Doctor of Philosophy (PhD)). University of Bath, UK, 2011).

[0141] A "reference expression level of a biological marker" is defined here as any expression level of that marker used as a reference. For example, a reference expression level can be obtained by measuring the expression level of the marker of interest in a child's skin model under specific conditions. Those skilled in the art will be able to choose these specific conditions according to their objective when implementing the procedure.

[0142] According to another example, the reference expression level of a biological marker is the expression level of said marker obtained in a child skin model, brought into contact with a reference formulation or active ingredient, and exposed to desiccation conditions.

[0143] When the reference expression level is an expression level obtained in a skin model exposed to desiccation conditions, those skilled in the art will readily understand that the desiccation conditions of the skin model used in the process of the invention and of the model used to obtain a reference expression level are preferably identical. Thus, the desiccation conditions used, in particular the atmospheric humidity, as well as the duration of exposure used in the process of the invention and of the model used to obtain a reference expression level, are preferably identical.

[0144] For example, a person skilled in the art may use as a reference formulation any formulation known in the prior art for its effect in the prevention or treatment of dry skin.

[0145] For illustrative purposes, the reference formulation is an oil-in-water emulsion, such as those described below. For example, the reference formulation is formulation A, which contains avocado perseoses. These are C7 sugars obtained from avocados. Avocado perseoses and their extraction processes are described in, among others, WO 2004 / 112742, WO 2005 / 105123, WO 2005 / 115421, WO 2008 / 025847, WO 2011 / 073281, WO 2014 / 017049, WO 2014 / 122326, WO 2015 / 044230, etc. PRODUCT A: EMULSION W / O type Cold Cream

[0146] MP % WATER QSP CAPRYLIC / CAPRIC TRIGLYCERIDE 5 à 20% COCO-CAPRYLATE / CAPRATE 1 à 10% POLYGLYCERYL-2-DIPOLYHYDROXYSTEARATE 1 à 10% GLYCERIN 1 à 10% WAX 1 à 10% VEGETABLE OIL 1 à 10% POLYGLYCERYL-3 DIISOSTEARATE 1 à 5% MAGNESIUM SULPHATE 0 à 2% STEARALKONIUM HECTORITE 0 à 2% PRESERVATIVES 0 à 2% PERSEA GRATISSIMA FRUIT EXTRACT / AVOCADO PERSEOSE 0 à 5% CERAMICS 0 à 1% PRODUCT B: W / O EMULSION type Cold Cream

[0147] MP % WATER QSP Paraffin oil 1 à 10% Glyceryl Stearate 1 à 10% Glycerin 1 à 10% Stearic acid 1 à 10% Cetyl alcohol 1 à 5% PEG-12 1 à 5% Triglycerides 1 à 5% Vegetable oils 1 à 5% Vegetable waxes 1 à 5% Octyl palmitate 0.5 à 5% Carbomer 0.5 à 2% Silicones 1 à 5% perfume 0.5 à 2% PRESERVATIVES 0 à 2% ACTIVE 0 à 5% PRODUCT C: EMULSION W / O type Cold Cream

[0148] MP % WATER QSP Thermal water 5 à 10% Paraffin oil 1 à 10% Cetyl palmitate 1 à 10% Glycerin 1 à 10% Glycols 1 à 10% Mineral waxes 1 à 10% Mineral oils 1 à 10% Vegetable waxes 1 à 5% MAGNESIUM SULPHATE 0 à 2% Magnesium stearate 0 à 2% Sorbitan isosterate 1 à 5% Polyglyceryl-3 polyricinoleate 0.5 à 3% PEG-30dipolyhydroxystearate 0.5 à 3% Silicones 0.5 à 2% PRESERVATIVES 0 à 2% ACTIFS 0 à 5% PRODUCT D: EMULSION W / O type Cold Cream

[0149] MP % AQUA QSP Eau thermale 5 à 10% Huile de paraffine 1 à 10% Glyceryl Stearate 1 à 10% Alcool cetylique 1 à 10% Cires minérales 1 à 10% C-20-40 Pareth 10 0.5 à 5% Cetyl phosphate 0.5 à 5% parfum 0.5 à 2% PRESERVATIVES 0 à 2% ACTIFS 0 à 5% PRODUCT E: COLD CREAM MILK

[0150] I NCI UE % INCI AQUA QSP CAPRYLIC / CAPRIC TRIGLYCERIDE 5 à 15% GLYCERIN 2 à 5% HELIANTHUS ANNUUS SEED OIL 2 à 5% HYDROGENATED COCONUT OIL 2 à 5% GLYCERYL STEARATE 1 à 3% CERA ALBA 1 à 3% 1,2-HEXANEDIOL 0.5 à 1.5% CETEARETH-20 0.5 à 1.5% PARFUM 0.5 à 1.5% GLYCERYL CAPRYLATE 0.1 à 0.5% ACRYLATES / C10-30 ALKYL ACRYLATE CROSSPOLYMER 0.1 à 0.5% CETEARETH-12 0.1 à 0.5% CETEARYL ALCOHOL 0.1 à 0.5% TOCOPHERYL ACETATE 0.1 à 0.5% SODIUM HYDROXIDE 0.05 à 0.3% PERSEA GRATISSIMA FRUIT EXTRACT 0.05 à 0.3% CERAMIDE NP 0.05 à 0.3% PHYTOSPHINGOSINE 0.05 à 0.3% 100.000000 Product F: Cold Cream Cleansing Gel

[0151] INCI UE % INCI AQUA QSP GLYCERIN 2 à 7% COCAMIDOPROPYL BETAINE 1 à 5% SODIUM MYRETH SULFATE 1 à 5% COCO-GLUCOSIDE 1 à 5% PEG-3 DISTEARATE 0.5 à 2% PEG-150 DISTEARATE 0.5 à 2% GLYCERYL CAPRYLATE 0.5 à 2% GLYCOL DISTEARATE 0.5 à 2% PARFUM 0.5 à 2% POLYQUATERNIUM-10 0.1 à 1% CERA ALBA 0.1 à 1% PRUNUS AMYGDALUS DULCIS OIL 0.1 à 1% CITRIC ACID 0.05 à 0.1% CERAMIDE NP 0.05 à 0.1% PERSEA GRATISSIMA FRUIT EXTRACT 0.05 à 0.1% 100.000000

[0152] According to another example, the reference expression level of a biological marker is the expression level of said marker obtained in a skin model obtained from a skin sample from a child, said model being untreated with the formulation or active ingredient of interest, and not exposed to desiccating conditions.

[0153] According to another example, the reference expression level of a biological marker is the expression level of said marker obtained in a skin model obtained from a skin sample from a child, said model being untreated with the formulation or active ingredient of interest, but exposed to desiccation conditions.

[0154] According to another example, the reference expression level of a biological marker is the expression level of said marker obtained in a skin model obtained from a skin sample from a child, treated with the formulation or active ingredient of interest, and not exposed to desiccating conditions.

[0155] Those skilled in the art will readily understand that the comparison in step (d) is preferably made between measurements of expression levels obtained for skin models derived from skin samples of children with similar, or even identical, histological structures. By "similar histological structures," we mean that the relative proportions of the cell types in the compared skin models are similar. Thus, it is preferable that the relative proportions of the cell types in the skin model in step (a) do not differ by more than 5% from the relative proportions of the cell types in the skin model used to obtain the reference expression level in step (d). By "relative proportion of a cell type," we mean the ratio of the number of cells corresponding to that cell type to the total number of cells in the skin model.Thus, for example, it is preferable that the proportion of keratinocytes to the total number of cells in the skin model from 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 in step d). "Identical histological structures" means that the relative proportions of the cell types in the compared skin models are identical. For example, the relative proportions of the cell types in the nipple skin model from step a) are identical to the relative proportions of the cell types in the skin model used to obtain the reference expression level in step d) when they do not differ by more than 0.1%.Advantageously, the proportion of keratinocytes to the total number of cells in the skin model of step a) does not differ by more than 0.1% from the proportion of keratinocytes to the total number of cells in the skin model used to obtain the reference expression level of step d).

[0156] A person skilled in the art will also readily understand that the comparison in step d) is preferably made between measurements of expression levels obtained for skin models that are of similar or even identical size, volume, or weight. Thus, it is preferable that the size, volume, or weight of the skin model in step a) does not differ by more than 5% from the size, volume, or weight of the skin model used to obtain the reference expression level in step d). For example, the size, volume, and weight of the skin model in 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 in step c).For illustration purposes, the size, volume and weight of the skin model in step a) do not differ by more than 0.1% from the size, volume and weight of the skin model used to obtain the reference expression level in step d).

[0157] Alternatively, if the skin models differ by more than 5% in size, volume and weight, a person skilled in the art may normalize the level obtained in step c) and the reference level in step d) using a normalization factor.

[0158] 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.

[0159] It can also be advantageous to use as a normalization factor the expression level of a gene that is expressed at the same level in all, or nearly all, cells of the organism. In other words, the expression level of a housekeeping gene is used, for example, as a normalization factor. As another example, the level obtained in step c) and the reference level in step d) are normalized using the expression level, not of the housekeeping genes themselves, but of the proteins they encode. A housekeeping gene is a gene expressed in all cell types that encodes a protein with 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). Housekeeping genes include, for example, the genes RPS28, GAPDH, B2M, TFRC, YWHAZ, RPLO, 18S, GUSB, UBC, TBP, GAPDH, PPIA, POLR2A, ACTB, PGK1, HPRT1, IPO8 and HMBS.

[0160] A person skilled in the art will thus be able to easily assess the effectiveness of the formulation of interest based on the comparison with step d).

[0161] According to an aspect not forming part of the invention, a kit for implementing a method according to the invention is described herein, comprising the means necessary for measuring the expression level of at least one marker selected from lipids, NMF, markers of skin inflammation, markers of barrier function, and markers preferentially expressed in stem cells. Preferably, the lipid is a ceramide, the marker of barrier function is selected from DSG, SCEL, PADI1, CASP14, LOR, TGM1, and CLDN1, and the marker preferentially expressed in stem cells is selected from ΔNp63, KRT15, ​​KRT19, BIRC5, and NOTCH1.

[0162] In particular, the kit also includes the means necessary to measure the expression level of a combination of biological markers chosen from the group comprising or consisting of: at least one marker of skin inflammation and at least one marker of the barrier as defined above; or at least one marker of skin inflammation and at least one marker preferentially expressed in stem cells, as defined above; or at least one marker of the barrier and at least one marker preferentially expressed in stem cells, as defined above.

[0163] As an example, said combination includes at least one marker of skin inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells, as defined above.

[0164] The following examples are provided here for illustrative purposes and are not, unless otherwise stated, intended to be exhaustive. DESCRIPTION OF THE FIGURES

[0165] There figure 1 shows the quantification of NMFs in the Normal Skin and Dry Skin panels, as well as the evolution of NMF quantities according to age groups. figure 2 shows the quantification of ceramides in the Normal Skin and Dry Skin panels, as well as the evolution of ceramide quantities according to age groups. EXAMPLES Example 1: Characterization of dry skin in babies and children in vivo

[0166] In order to characterize dry skin in babies and children, an exploratory clinical study was carried out. 1. Matériel et méthodes

[0167] A clinical study under pediatric and dermatological control was conducted on two panels (Normal Skin panel and Dry Skin panel; classification after clinical examination carried out by the investigator) of 40 subjects each, divided into 4 age groups: Group 1: 1-28 days; Group 2: 3-6 months; Group 3: 1 year; Group 4: 2-4 years (respectively Gpe 1, Gpe 2, Gpe 3, Gpe 4).

[0168] Biological samples were collected from the subjects' forearms using swabs. The levels of NMFs (Natural Moisture Factors) and ceramides were measured in the collected samples using liquid chromatography coupled with mass spectrometry (LC / MS) for ceramides and liquid chromatography coupled with UV detection (LC / UV) for NMFs. Analyse des céramides

[0169] The presence of ceramides with a sphingosine [S], dihydrosphingosine [DS] and phytosphingosine [P] type sphingosine base with an even chain length of 16 to 22 carbon atoms was investigated by an LC / MS method.

[0170] The ceramide content was normalized by the amount of total protein (BCA assay). Analyse des éléments du NMF

[0171] Filaggrin catabolites were quantified by an LC / UV method allowing screening of urocanic acid (UCA) under its two isomers (cis and trans) as well as L-pyrrolidone carboxylic acid (PCA).

[0172] The NMF content was normalized by the amount of total protein (BCA assay). 2. Résultats et conclusion

[0173] The results presented below show that subjects in the Dry Skin panel have significantly lower NMF levels than those in the Normal Skin panel, regardless of age group ( figure 1 , Table 1). These observations show that low levels of NMFs constitute a biochemical marker of dry skin in newborns, infants and young children.

[0174] On the other hand, the quantities of ceramides are lower for subjects in the Dry Skin panel than for subjects in the Normal Skin panel ( figure 2 , table 1), showing that ceramides are also a marker of dry skin condition in newborns, infants, and children with clinically dry skin.

[0175] In conclusion, the amount of NMFs and ceramides is decreased in dry children's skin compared to normal children's skin. Tableau 1 Overall NMF and ceramide content across all Normal and Dry Skin panels - Student's t-test Peau Normale Peau Sèche Comparison of Dry Skin vs. Normal Skin Sum of NMFs (µg / mg of protein) 97,37 89,43 -8,2% p<0,05 Total Ceramides (µg / 100mg of protein) 92,68 84,44 -8,9% p<0,05 Example 2: Model validation in vitro of "Dry Skin"

[0176] In order to validate the modeling of a dry skin phenotype, the production of NMF (Natural Moisturizing Factor) and ceramides by epidermis incubated in a dry atmosphere was evaluated. 1. Materials and methods

[0177] The reconstructed epidermis was made using keratinocytes from a 1-year-old donor.

[0178] Epidermal reconstruction was performed according to the model derived from the method of Poumay et al (Arch Dermatol Res 2004; 296: 203-11). After 2 days of immersion culture, the reconstructed human epidermis (RHE) was cultured at the air / liquid interface for 11 days.

[0179] On day 11, the epidermis was incubated for 48 hours in a humid incubator for the control epidermis (normal condition: 37°C, 5% CO2 and relative humidity >99%) or in a dry incubator (37°C, 5% CO2 and relative humidity <25%).

[0180] At the end of the incubation period, the quantities of NMF and ceramides produced by the epidermis were evaluated.

[0181] The experiments were repeated 3 times; for each of these 3 trials, 3 replicates were carried out and analyzed. Ceramide analysis

[0182] Epidermal lipids were extracted by agitating the epidermis with a mixture of organic solvents for 2 hours at room temperature. A solid / liquid extraction process was then performed to isolate the ceramides from the other lipids constituting the epidermis.

[0183] Ceramide levels were analyzed by LC / MS as described in Example 1. Analysis of the elements of the NMF

[0184] The reconstructed epidermis was extracted under agitation for 2 hours at room temperature from an aqueous mixture in the presence of a non-ionic surfactant to promote the extraction of markers of interest.

[0185] MNF levels were analyzed by LC / UV as described in Example 1. 2. Results and conclusion

[0186] Ceramides are important structural components of the epidermis. They are one of the constituents of the lipid matrix of the stratum corneum. The role of this matrix is ​​crucial in regulating water permeability. It acts as a hydrophobic barrier that regulates water flow through the subcutaneous tissue. The lipid matrix is ​​composed of an equimolar mixture of ceramides (45-50% of the total weight), cholesterol (20-25%), and free fatty acids (10-15%). Ceramides are derived from the transformation of sphingomyelin by sphingomyelinases and of glucoceramides by β-glucocerebrosidase.

[0187] The main function of the NMF is to maintain an optimal water level in the stratum corneum. A well-known source of NMF constituents is filaggrin.

[0188] Incubation of reconstructed epidermis for 1 year in a dry oven induced a significant inhibition of ceramide and NMF content in the epidermis (Table 2).

[0189] The decrease in the amount of NMF and ceramides is one of the main characteristics of dry skin, well-known and described in the literature. Furthermore, Example 1 shows that the amount of NMF and ceramides also decreases in children's dry skin. The established model, consisting of incubating reconstructed epidermis in a dry atmosphere, is therefore highly representative of the observed phenotype of dry skin in children. in vivo. Table 2 : NMF and ceramide content in 1-year-old reconstructed epidermis incubated under normal conditions (control) or in a dry incubator (dry atmosphere) Controlled skin Epidermis in a dry atmosphere Inhibition Significance (Student's t-test) NMF content (µg / mg of protein) 17,05±0,21 12,44±0,82 -27% P<0,001 Ceramide content (a / mg of protein) 173,6±3,86 149,4±2,62 -14% P<0,001 Example 3: Evaluation of the effect of delipidation on the quantity of ceramides in reconstructed epidermis

[0190] In order to determine if other models in vitroDry skin models allow for the reproduction of the phenotype of dry skin in children; the dry skin induction model by delipidation was tested. The quantity of ceramides, one of the specific markers of dry skin in children, was analyzed in this model.

[0191] Reconstructed epidermis was produced according to the model derived from the method of Poumay et al (Arch Dermatol Res 2004; 296: 203-11). After 2 days of immersion culture, the reconstructed human epidermis (RHE) was cultured at the air / liquid interface for 10 days.

[0192] On day 10, the epidermis was treated by topical application of a 30% 1:1 ether / acetone mixture to induce alteration of the barrier by delipidation, then incubated for 48 hours (Miyamoto et al., 2002, Akiyama et al., 2010, Valvetcha et al., 2015).

[0193] At the end of the incubation period, the quantities of ceramides produced by the epidermis were evaluated. Ceramide analysis

[0194] Epidermal lipids were extracted by agitating the epidermis with a mixture of organic solvents for 2 hours at room temperature. A solid / liquid extraction process was then performed to isolate the ceramides from the other lipids constituting the epidermis.

[0195] The presence of ceramides with a sphingosine [S], dihydrosphingosine [DS] and phytosphingosine [P] type sphingosine base with an even chain length of 16 to 22 carbon atoms was investigated by an LC / MS method. Results and conclusion

[0196] Lipid removal from the epidermis by ether / acetone treatment does not induce a decrease in the amount of ceramides produced by the epidermis (Table 3).

[0197] Thus, surprisingly, delipidation does not allow for modeling the demonstrated dry skin phenotype in vivo.Only the "dry skin" model obtained by incubation in a dry atmosphere oven, designed by the present Inventors and described in Example 2 above, makes it possible to reproduce the observed phenotype in vivo for children's dry skin. Table 3: Overall ceramide content for control and lipid-depleted epidermis - Student's t-test Epidermis control Lipid-depleted epidermis Comparison of delipidated epidermis vs. control epidermis Ceramides (Arbitrary unit) 234,3 ± 26,0 227,9 ± 3,9 -2.8% ns Example 4: Characterization of the response of infant epidermis compared to adult epidermis 1. Materials and methods

[0198] Reconstructed epidermis was produced, as described above, with keratinocytes from a 1-year-old donor on the one hand and with keratinocytes from a 19-year-old donor on the other hand; the keratinocytes from both donors came from a preputial sampling.

[0199] On day 10, the epidermis was incubated for 24 to 48 hours in a humid incubator for the control epidermis (normal condition: 37°C, 5% CO2 and relative humidity >99%) or in a dry incubator (37°C, 5% CO2 and relative humidity <25%).

[0200] After 24 hours of incubation, gene expression of markers of inflammation, hydration, barrier function and stem cells was assessed by qRT-PCR (quantitative real-time PCR).

[0201] After 48 hours of incubation, the viability and morphology of the epidermis were studied. Evaluation of the viability of reconstructed epidermis

[0202] The viability of the epidermis was assessed using a 2x MTT reduction test: the epidermis was incubated in the presence of MTT (tetrazolium salt), the transformation of which into blue formazan crystals is proportional to the activity of succinate dehydrogenase (a mitochondrial enzyme). After cell dissociation and solubilization of the formazan by the addition of isopropanol / HCl, the optical density (OD), representative of the number of viable cells and their metabolic activity, was measured at 540 nm. Evaluation of the morphology of reconstructed epidermis

[0203] The morphology of the reconstructed epidermis was evaluated in n=2 by histological analysis after Hematoxylin / Eosin staining.

[0204] The epidermis was fixed and embedded in paraffin, and cross-sections were prepared using a microtome. The sections were deparaffinized and then stained with hematoxylin and eosin. Hematoxylin stains cell nuclei blue / violet; eosin stains cytoplasm a more or less vivid pink.

[0205] Analysis of histological sections by microscopy made it possible to evaluate and quantify certain morphological parameters such as the thickness of the layer of living cells. Analysis of differential gene expression

[0206] Marker expression was assessed by qRT-PCR on messenger RNAs extracted from RHEs of each treatment.

[0207] Gene expression analysis was performed in n=2 using an array PCR containing 30 genes of interest and 2 housekeeping genes. Total RNA from each sample was extracted using TriPure Isolation Reagent® according to the manufacturer's recommended protocol. RNA quantity and quality were assessed by capillary electrophoresis (Bioanalyzer, Agilent). Complementary DNA (cDNA) was synthesized by reverse transcription of the RNA in the presence of oligo(dT) and the enzyme reverse transcriptase. The resulting cDNA was quantified by spectrophotometry, and the cDNA quantities were then adjusted.

[0208] PCR reactions were performed using quantitative PCR with a light cycler system (Roche Molecular Systems Inc.) according to the manufacturer's recommended procedure. The reaction mixture for each sample consisted of: cDNA, primers for the various markers used, a reaction mixture containing the enzyme taq DNA polymerase, the marker SYBR Green I (a DNA intercalating agent), and MgCl2.

[0209] The incorporation of fluorescence into the amplified DNA is measured continuously during PCR cycles.

[0210] Quantitative analysis of the results is based on the collection of threshold cycles (or Ct). The threshold cycle corresponds to the point at which the fluorescence emission signal is statistically and significantly higher than the background noise. The threshold cycle is directly correlated with the number of initial copies of the target DNA.

[0211] Table 4 lists the genes that were studied.

[0212] The relative gene expression (RE) value is expressed in arbitrary units according to the following formula: ER = 1 / 2 nombre de cycles × 10 6 Table 4 Classification and names of the genes studied Cluster name Abbreviation Gene name Housekeeping RPS28 Ribosomal protein 28S GADPH Glyceraldehyde-3-phosphate dehydrogenase Nonspecific and neurogenic inflammation IL1A Interleukin 1 alpha IL8 Interleukin 8 PTGS2 Prostaglandin synthase 2 NGFR Nerve Growth Factor Receptor PLA2G2F Phospholipase A2 Group IIF TAC1 Tachykinin precursor 1, or Substance P TAC1R Tachykinin receptor 1, or Substance P receptor TRPV1 Transient receptor potential cation channel, subfamily V, member 1 TRPV3 Transient receptor potential cation channel, subfamily V, member 3 MRGPRD MAS related GPR family member D F2RL1 Coagulation factor II (thrombin) receptor-like 1 or Proteinase Activated Receptor 2 (PAR2) OSMR Oncostatin M receptor, interleukin 31 receptor Epidermal differentiation, barrier function, hydration DSG Desmoglein 1 SCEL Skyline PADI1 Peptidyl Arginine Deiminase 1 CASP14 Caspase 14 LOR Loricrine TGM1 Transglutaminase 1 CLDN1 Claudine 1 Stem cells TP63 Tumor Protein P63 KRT15 Keratin15 KRT19 Keratin 19 BIRC5 Baculoviral IAP repeat-containing 5 or surviving (=Survivin) NOTCH1 Notch Homolog 1 2. Results a. Morphology and viability of epidermis

[0213] Incubation in a dry atmosphere did not significantly alter the morphology of the epidermis observed under the microscope after hematoxylin / eosin staining (data not shown); moreover, epidermal viability was impacted similarly in 1-year-old and adult epidermis (30% and 29% inhibition, respectively, Table 5). However, the measurement of the thickness of the living cell layer was more strongly impacted in 1-year-old epidermis (-18%) compared to adult epidermis, in which no decrease was observed for this parameter (Table 5).

[0214] These observations tend to show an increased sensitivity of 1-year-old skin to dehydrating stress. Table 5: Analysis of the viability and thickness of the living cell layer Epidermis Control Dry atmosphere Viability (MTT) 1 year 100% 70% Adult 100% 71% Thickness of the layer of living cells 1 year 100% 82% Adult 100% 100% b. Analysis of the expression of marker genes of nonspecific and neurogenic inflammation

[0215] Incubation in a dry atmosphere induced an increase in the level of expression of nonspecific and neurogenic inflammation markers, both in 1-year-old reconstructed epidermis and in adult reconstructed epidermis (Table 6).

[0216] This stimulation is however much more important in the case of reconstructed infant epidermis (+355%) compared to adult epidermis (+114%).

[0217] This suggests increased sensitivity of infant skin, whose inflammatory and neuro-sensory response is exacerbated in response to dehydrating stress. Table 6 : Gene expression level of inflammation markers (relative expression in % compared to the control cultured under normal conditions) Epidermis 1 year checkups Epidermis 1 year in a dry atmosphere Controlled adult epidermis Adult epidermis in a dry atmosphere IL1A 100 177 100 155 IL8 100 314 100 147 PTGS2 100 1421 100 666 NGFR 100 223 100 100 TRPV1 100 675,5 100 125 TRPV3 100 606 100 150 MRGPRD 100 246 100 351 F2RL1 100 171 100 119 OSMR 100 263 100 113 Average expression of inflammation markers (%) 100 455 100 214 Increase compared to the control (%) - +355% - +114% c. Analysis of stem cell marker gene expression

[0218] Stem cells of constantly renewing tissues are classically defined as rare and relatively quiescent cells. They possess a unique capacity for self-renewal and tissue regeneration, enabling them to maintain homeostasis and the integrity of the tissue in which they reside.

[0219] Among epidermal stem cells, interfollicular stem cells located in the basal layer constitute the main epidermal stem cell reservoir. These cells reside in an anatomical and functional microenvironment, the niche, which helps maintain their characteristics, particularly when physiological conditions change. Interfollicular stem cells and their niches are involved in maintaining epidermal integrity and regeneration. Stem cells can only be identified by tracking several markers. We therefore evaluated the expression levels of various gene markers characteristic of stem cells in a "dry skin" model.

[0220] Incubation in a dry atmosphere induced a significant decrease (-45%) in the mean expression level of the studied stem cell marker pool (Table 7) in 1-year-old epidermis, whereas no inhibition of the expression of these markers was observed in adult epidermis (+12%).

[0221] This tends to show an increased vulnerability of this cellular capital in 1-year-old epidermis. Table 7: Gene expression level of stem cell markers (relative expression in % compared to the 1-year control) Epidermis 1 year checkups Epidermis 1 year in a dry atmosphere Controlled adult epidermis Adult epidermis in a dry atmosphere KRT19 100 65 84 120 BIRC5 100 64 85 94 TP63 100 13 86 80 NOTCH1 100 77 45 43 Average expression of the stem cell marker pool (%) 100 55 75 84 Modulation in relation to control (%) - -45% - +12% d. Analysis of the expression of barrier and hydration marker genes

[0222] Incubation in a dry atmosphere induced a decrease in the level of expression of barrier markers, whether in 1-year-old reconstructed epidermis or in adult reconstructed epidermis (Table 8).

[0223] This inhibition is however more significant in the case of reconstructed infant epidermis (-57%) compared to adult epidermis (-38%).

[0224] This suggests a greater vulnerability of barrier function markers in infant epidermis. Table 8: Gene expression levels of barrier and hydration markers (relative expression in % compared to the 1-year control) Epidermis 1 year checkups Epidermis 1 year in a dry atmosphere Controlled adult epidermis Adult epidermis in a dry atmosphere DSG1 100 56 153 92 SCEL 100 20 62 34 PADI1 100 51 117 78 CASP14 100 47 112 74 Average expression of barrier markers (%) 100 43 111 69 Inhibition compared to control (%) - -57% - -38%

[0225] There desmoglein 1 (DSG1) is a constituent protein of corneodesmosomes which ensure the cohesiveness of corneocytes within the stratum corneum.

[0226] There skyline (SCL) is a precursor of the horny envelope.

[0227] There PADI1 (Peptidyl arginine Deiminase 1) and caspase 14 (CASP14) are two enzymes involved in the processing of filaggrin to obtain NMF.3. Conclusion

[0228] Comparative analysis of the behavior of reconstructed 1-year-old and adult (19-year-old) epidermis in the dry skin induction model shows a greater vulnerability and susceptibility of infant epidermis to dehydrating stress induced by incubation in a dry atmosphere.

[0229] This increased vulnerability justifies the development of cosmetic products for dry skin specifically adapted to baby's skin. Example 5: Evaluation of products for dry skin

[0230] The previously established infant dry skin model, consisting of incubating reconstructed 1-year-old infant epidermis in a dry atmosphere, was used to comparatively evaluate the biological efficacy of 4 cosmetic products intended for dry skin. 1. Materials and methods

[0231] Reconstructed epidermis was produced, as previously described, from keratinocytes of a 1-year-old donor.

[0232] On day 11, the epidermis was treated by topical application of the test products at a rate of 5 mg / cm² and then incubated for 6h, 24h or 48h in a dry oven (dry atmosphere condition: 37°C, 5% CO2 and relative humidity <25%), except for the control epidermis in a humid oven (37°C, 5% CO2 and relative humidity >99%) and analyzed as described above: After 6h and 24h of incubation, the gene expression of markers of nonspecific and neurogenic inflammation, hydration, barrier function and stem cells (Table 1) was evaluated by qRT-PCR (quantitative real-time PCR) in n=2.

[0233] After 48 hours of incubation, the viability (MTT) and morphology (histology, hematoxylin / eosin staining) of the epidermis were studied in n=2 and the production of NMF and ceramides analyzed in n=3.

[0234] Products being tested =reference formulations as cited above: Product A Product B Product C Product D 2. Results a. Morphology and viability of epidermis

[0235] Just as in the previous stage of model development, incubation in a dry atmosphere did not significantly alter the morphology and viability of the epidermis (Table 9). Table 9 Analysis of epidermal viability Viability (MTT) Epidermis control 100% Dry oven indicator 87% Product A 90% Product B 89% Product C 75% Product D 97% b. Analysis of the expression of marker genes for neurogenic and nonspecific inflammation

[0236] Products A, B, and C inhibit inflammation markers expressed under dry incubation conditions (Table 10). These products therefore offer protection against skin reactivity, which can be exacerbated under these conditions. Only product D has no inhibitory effect on these markers. Table 10 : Gene expression levels of neurogenic and nonspecific inflammation markers (relative expression in % compared to the dry oven control) PLA2G2F TAC1 TAC1R Average marker expression (%) Inhibition compared to the dry oven control Dry oven indicator 100 100 100 100 Product A 90,5 81,5 70 81 -19% Product B 75,5 66 55 65 -35% Product C 103,5 75,5 75 85 -15% Product D 87,5 95,5 105,5 96 -4% c. Analysis of stem cell marker gene expression

[0237] Only product A completely restores (96% restoration) the level of expression of stem cell markers inhibited by incubation in a dry atmosphere with an efficacy superior to the other products tested (Table 11).

[0238] Product A therefore protects the cellular capital in the dry skin model. Table 11 : Gene expression level of stem cell markers (relative expression in % compared to the 1-year control) KRT15 KRT19 BIRC5 TP63 Average expression of the stem cell marker pool (%) % restoration Control 100 100 100 100 100 Dry oven indicator 25 74 45 47 48 Product A 97 130 78 90 98 96% Product B 80 68 32 62 61 61% Product C 86 131 21 70 77 77% Product D 86 74 19 71 63 63% d. Analysis of the expression of barrier marker genes

[0239] Under dry incubation conditions, products A and B restored the level of expression of barrier marker genes and showed a higher restoration efficiency (140% and 113% respectively) than the other formulas tested (Table 12).

[0240] Thus, product A, and to a lesser extent product B, protect the expression of barrier markers altered by incubation in a dry atmosphere. Table 12 : Gene expression level of barrier markers (relative expression in % compared to the 1-year control) LOR TGM1 CLDN1 DSG1 Average expression of barrier markers (%) % restoration Control 100 100 100 100 100 Dry oven indicator 88 93 93 68 85 Product A 103 109 111 103 106 140 Product B 97 109 110 91 102 113 Product C 78 91 92 114 94 60 Product D 72 87 86 101 87 13

[0241] There loricrine (LOR) is a late marker of epidermal differentiation involved in the formation of the horny envelope.

[0242] There transglutaminase 1 (TGM1) enzyme responsible for the cross-linking of different proteins for the formation of the corneal envelope, ensuring the seal / solidity of the stratum corneum.

[0243] There Claudine 1 (CLDN1) is one of the constituent proteins of tight junctions, involved in the skin barrier function, notably controlling water flow.

[0244] There desmoglein 1 (DSG1) is a constituent protein of corneodesmosomes which ensure the cohesiveness of corneocytes within the stratum corneum.

[0245] There corneodesmosine is a constituent protein of corneodesmosomes, junctions which form between corneocytes to maintain the structure of the stratum corneum and thus participate in good desquamation of the skin

[10] . e. Analysis of NMF and ceramide production

[0246] Products A and B restored the production of inhibited ceramides and NMF in the dry skin model (Table 13), while the other two products tested (C and D) showed less efficacy: virtually no effect on ceramide production, slight increase in NMF production. Table 13 : NMF and ceramide content in 1-year-old reconstructed epidermis incubated under normal conditions (control) or in a dry incubator (dry atmosphere) NMF content (µg / mg of protein) Ceramide content (a / mg of protein) Control 16,6±1,8 179,5 Dry oven indicator 11,7±8,8 -30% 138,25 -23% Product A 17,3±3,6 +48% 178,25 +24% Product B 18,3±8,1 +56% 183,25 +27% Product C 13,9±2 +19% 127,3 -12% Product D 14,5±1,7 +24% 150,95 +5%

Claims

1. Method for evaluating the in vitro efficacy of an active ingredient or a formulation on preventing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) contacting said active ingredient or said formulation with a reconstructed skin model, said model being obtained from a skin sample from a child; b) growing the reconstructed skin model from step a) under drying conditions; c) measuring the expression level of a combination of biological markers in the skin model of step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) evaluating the efficacy of said active or formulation as a function of the level of step c).

2. Method for evaluating the in vitro efficacy of an active ingredient or a formulation in reducing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) growing the reconstructed skin model under drying conditions, said model being obtained from a skin sample from a child; b) contacting said active ingredient or formulation with the reconstructed skin model from step a); c) measuring the expression level of a combination of biological markers in the skin model of step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one barrier marker and at least one marker preferentially expressed in stem cells; and d) evaluating the efficacy of said active or formulation as a function of the level of step c).

3. Method for identifying an active ingredient or a formulation for preventing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) contacting a candidate active or formulation with a reconstructed skin model, said model being obtained from a skin sample from a child; b) growing the reconstructed skin model from step a) under drying conditions; c) measuring the expression level of a combination of biological markers in the skin model of step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) determining whether said candidate active or formulation is a formulation or active for preventing the effects of dehydration on children's skin as a function of the level of step c).

4. Method for identifying an active ingredient or a formulation for reducing the effects of dehydration on children's skin, characterized in that said method comprises the following steps: a) growing the reconstructed skin model under drying conditions, said model being obtained from a skin sample from a child; b) contacting a candidate active or formulation with the skin model from step a); c) measuring the expression level of a combination of biological markers in the skin model of step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) determining whether said candidate active or formulation is a formulation or active for reducing the effects of dehydration on children's skin as a function of the level of step c).

5. Method for evaluating the tolerance of an active ingredient or a formulation on dehydrated children's skin, said method comprising the following steps: a) contacting said active ingredient or said formulation with a reconstructed skin model, said model being obtained from a skin sample from a child; b) growing the reconstructed skin model from step a) under drying conditions in the presence of the active ingredient or formulation; c) measuring the expression level of a combination of biological markers in the skin model of step b), said combination comprising at least one lipid and NMF and at least one marker of inflammation and at least one marker of the barrier and at least one marker preferentially expressed in stem cells; and d) determining whether said active agent, active or formulation is well tolerated by the child's skin as a function of the level of step c).

6. Method according to any one of claims 1 to 5, characterized in that the lipid is a ceramide, the inflammation marker is selected from IL1A, IL8, PTGS2, NGFR, TAC1, TAC1R, TRPV1, TRPV3, MRGPRD, OSMR, PLA2G2F and F2RL1, the barrier function marker is chosen from DSG, SCEL, PADI1, CASP14, LOR, TGM1 and CLDN1, and the marker preferentially expressed in stem cells is chosen from ΔNp63, KRT15, KRT19, BIRC5, and NOTCH1.

7. Method according to any one of claims 1 to 6, characterized in that the skin sample comes from a donor chosen from the group consisting of newborns, whose age is between 0 and 1 month, infants, whose age is between 1 month and 2 years, and children whose age is between 2 years and 16 years.

8. Method according to any one of claims 1 to 7, characterized in that the skin sample comes from skin with phototype I, II, III, IV, V or VI.

9. Method according to any one of claims 1 to 8, characterized in that the reconstructed skin model is selected from suspension skin cell cultures, monolayer skin cell cultures, bilayer skin cell cultures, reconstructed skin cultures and reconstructed mucosa cultures.

10. Method according to claim 9, characterized in that the cells of said model are derived from a skin tissue explant or from stem cells differentiated into skin cells.

11. Method according to any one of claims 7 to 9, characterized in that said model comprises at least fibroblasts or keratinocytes.