WO2012059703A1 - In vitro or ex vivo method for storing and/or keeping an epidermis alive - Google Patents
In vitro or ex vivo method for storing and/or keeping an epidermis alive Download PDFInfo
- Publication number
- WO2012059703A1 WO2012059703A1 PCT/FR2011/052587 FR2011052587W WO2012059703A1 WO 2012059703 A1 WO2012059703 A1 WO 2012059703A1 FR 2011052587 W FR2011052587 W FR 2011052587W WO 2012059703 A1 WO2012059703 A1 WO 2012059703A1
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- WO
- WIPO (PCT)
- Prior art keywords
- epidermis
- skin
- fragment
- solution
- liquid matrix
- Prior art date
Links
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/128—Chemically defined matrices for immobilising, holding or storing living parts, e.g. alginate gels; Chemically altering living parts, e.g. by cross-linking
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0629—Keratinocytes; Whole skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
- C12N5/0698—Skin equivalents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
- C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1323—Adult fibroblasts
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to an in vitro or ex vivo process for the preservation and / or survival maintenance of an epidermis.
- the present invention includes the epidermal model thus obtained and its use in a kit or method for screening or selecting cosmetic or pharmaceutical compounds.
- the epidermis is a squamous and squamous squamous epithelium. It forms a barrier that resists the ravages of desiccation, as well as mechanical, chemical and microbial attacks.
- the main cell type constituting the epidemic is the keratinocyte. This tissue also includes other cell populations such as melanocytes, Langerhans cells, and Merkel cells.
- the epidermis is conventionally subdivided into four distinct layers comprising inner layers towards the most superficial layers: the basal layer (one layer), the spinous layer (4-15 layers), the granular layer (1-3 layers) and the stratum corneum (5-10 layers).
- the epidermis rests on the dermis thanks to a basement membrane composed among others of collagen molecules.
- the dermis contains very dense vascular and nervous networks as well as epidermal appendages, keratinized structures extending the epidermis and including hair follicles, sebaceous glands, and sweat glands.
- the reconstructed skin or epidermis models described in these documents consist of human keratinocytes deposited on a support, often a dermis equivalent, and cultured under conditions such that they enter into a differentiation program resulting in to the formation of an epidermis equivalent.
- Reconstructed epidermal or skin models are frequently used for dermatology investigations in substance testing, as for drug candidates or developing cosmetic actives.
- the pharmacological effects of these molecules, in particular their corrosive potential or the irritant, are also analyzed thanks to these systems.
- the reconstructed epidermis are also useful for answering questions of the molecular, immunological or histological type. This includes penetration and absorption studies. In comparison with preclinical studies using animals and clinical studies in humans, reconstructed epidermis / skins allow results to be obtained more quickly and in a more reproducible way.
- melanocytes or immune cells can be added to the keratinocyte cultures (Céline AUXENFANS et al, "Evolution of three dimensional skin equivalents models reconstructed in vitro by tissue engineering", Eur J Dermatol 2009 19 (2): 107-13).
- the technologies used do not yet allow the production of epidermis containing both keratinocytes, melanocytes, Langerhans cells and Merkel cells.
- many technological barriers persist to reconstruct in vitro epidermal appendages such as hair follicles, sebaceous glands and sweat glands.
- the applicant has demonstrated that the implementation of the method of the invention as described below, in particular comprising the deposition of skin fragment in a first step on a liquid phase, then, in a second stage, on a liquid phase which can induce solidification, allowed to obtain preservation or survival maintenance of an explant epidermis that can be used as an in vitro model or ex vivo.
- the subject of the present invention is therefore an in vitro or ex vivo method for the preservation or survival maintenance of an explant epidermis.
- the invention particularly includes a method for ex vivo survival of an entire epidermis explant with these appendices.
- said skin fragment is a fragment of mammalian skin, preferably human skin.
- the present invention also relates to an epidermis, preferably isolated, obtainable or directly obtained by such a method and its use as a model for evaluating ex vivo or in vitro ef and cosmetic, pharmaceutical or dermatological products to topical administration.
- the present invention relates to an ex vivo or in vitro method of preservation or maintenance in survival of an epidermis from a skin fragment comprising epidermis and dermis, this fragment having been previously taken from a mammal, characterized in that said method comprises the following steps:
- This method is advantageous in that it makes it possible to obtain an epidermis model that can be preserved or maintained in prolonged survival without appreciable degradation.
- said epidermis comprises all of its cell layers as well as the epidermal appendices in their entirety.
- set of its cell layers for an epidermis is meant here the basal layer, the spinous layer, the granular layer and the stratum corneum.
- epidermal appendices in their entirety is meant here hair follicles, sebaceous glands and sweat glands.
- said at least one protease contained in the enzyme solution is a protease whose activity makes it possible to separate the epidermis from the dermis, in particular by degradation of the basement membrane proteins separating the epidermis from the dermis.
- said protease is chosen from the family of dispases, trypsins or thermolysins.
- the level of said enzymatic solution in the container (depth) is greater than the thickness of the sampled fragment.
- the enzymatic solution is an aqueous solution, preferably physiological, preferably compatible with the preservation and / or maintenance in ex vivo or in vitro survival of the epidermis, such as for example a phosphate buffered saline (PBS), a Hank's balanced solution (HBSS) or any other buffer or physiological solution, such as a 0.9% aqueous NaCl solution (V / V), compatible with the preservation and / or maintenance in survival and / or cell culture of mammal.
- PBS phosphate buffered saline
- HBSS Hank's balanced solution
- V / V 0.9% aqueous NaCl solution
- cell culture is meant here in particular the maintenance of the physiological state of the cell.
- step c) of the process of the invention said detachment is induced by the proteases contained in the enzymatic solution.
- step c) of the process of the invention the dermis thus detached progressively dips into the bottom of the container.
- the invention particularly comprises a method according to the invention for preserving and / or surviving ex vivo or in vitro an entire explant of epidemic with these appendices, said method comprising the following steps:
- the invention relates to the use of such an epidermis obtained by a method according to the present invention for model ex vivo or in vivo, particularly for toxicological analyzes or for research purposes.
- said toxicological analyzes are chosen from sensitization, absorption, metabolism, corrosion, or irritation tests on the skin, preferably the epidermis.
- said method comprises in step e): i) the removal of the epidermis resting on the enzymatic solution and its deposition on said liquid matrix capable of being able to solidify, this liquid matrix being contained in a second container; or
- said method comprises in step e):
- said solidifying liquid matrix further contains cells other than cells of the epidermis, preferably selected from the group of cells consisting of fibroblasts, endothelial cells, nerve cells, cells constituting the dermis and hypodermis and immune cells; or
- replacing the matrix containing the enzymatic solution with a liquid matrix in the initial container said replacement liquid matrix being a liquid matrix capable of being able to solidify, and, where appropriate, further containing cells other than epidermis, preferably selected from the group of cells consisting of fibroblasts, endothelial cells, nerve cells, cells constituting the dermis and hypoderm and immune cells.
- step e) of said process the fragment obtained is then placed on a liquid matrix capable of being able to solidify said liquid matrix capable of supplying all the nutritive and / or necessary ingredients for its cultivation, in particular to the maintenance of the state of initial physiology of the cells which constitute it.
- step e) of said method the fragment obtained is then placed on a liquid matrix capable of being able to solidify said liquid matrix that can additionally provide all the ingredients and all the cells necessary for the preparation of a complete skin equivalent reproducing the characteristics of a skin in vivo.
- said liquid matrix does not cover the upper face of the epidermis before this matrix is solidified.
- the present invention relates to an ex vivo or in vitro method of preservation or maintenance in survival of an epidermis from a skin fragment, said method comprises the following steps:
- step B) the culture of the epidermis thus obtained in step A) under suitable culture conditions, preferably, said epidermis being in contact with the air.
- the process according to the invention is characterized in that said epidermis is an entire epidermis with its appendages, comprising the hair follicles, the sebaceous and sweat glands.
- said fragment previously removed is a fragment of cylindrical shape, preferably whose size is between 1 to 10 mm in diameter, more preferably from 1 to 8 mm and from 0.5 to 8 mm thickness, preferably 1 to 5 mm.
- the enzymatic solution able to degrade the proteins of the basement membrane separating the epidermis from the dermis and contained in the said matrix in step a) is a solution containing a protease, preferably dispase. , trypsin or thermolysin, preferably at a concentration of between 1 and 20 mg / ml (V / V) of solution, more preferably between 5 and 15 mg / ml and between 7.5 and 12.5. mg / ml solution.
- said liquid matrix capable of being able to solidify under the action of an increase in temperature or of a specific compound or composition is chosen from any liquid solution, preferably nutritive, capable of solidifying or gelling under particular conditions compatible with the survival and culture of epidemic cells, preferably chosen from blood plasma or a solution derived from blood plasma, in particular diluted in physiological buffer to at most 10%, preferably at least 20%, at least 30% and 40%, a fibrinogen solution, a collagen solution, gelatin, natural synthetic polymeric gels such as agarose, starch or polysaccharide gels.
- said liquid matrix capable of being able to solidify is a liquid solution derived from blood plasma treated with an anticoagulant agent with reversible properties, preferably with sodium citrate;
- the solidification of said matrix is obtained in the presence of calcium ions, preferably also in the presence of thrombin.
- step c) said skin fragment thus disposed on the solution is incubated for a period of at least 10 hours, preferably at 4 ° C., of more preferably overnight at 4 ° C.
- step e) said liquid matrix is solidified after a maximum of 8 hours, preferably less than 2 hours or less than one hour, a duration of less than 30 min, preferably less than 10 min being the most preferred time to initiate the solidification phase of the liquid matrix.
- said liquid matrix capable of being able to solidify is a solution derived from blood plasma containing from 25% to 60%, preferably between
- said solidifying liquid matrix is a liquid solution of fibrinogen and thrombin, or collagen and for which the incubation at 37.degree. ° C allows its solidification.
- the present invention relates to an epidermal model that can be obtained or directly obtained by the method according to the present invention.
- a further object is also a kit comprising such an epidermis model resting in particular on a solid and, where appropriate, nutritive matrix, this solid matrix being adapted by its composition to the preservation and / or the maintenance in survival of said epidermis, where appropriate to the multiplication and differentiation of the cells that may be included in this solid matrix.
- the subject of the invention is also a kit for evaluation of a cosmetic, dermatological or therapeutic compound for the skin comprising a skin or epidermis model obtained according to the method of the invention.
- Another subject of the present invention relates to a method for in vitro screening of candidate compounds for the cosmetic or therapeutic treatment of the skin, preferably of the epidermis, preferably of the epidermis associated with the dermis, comprising the following steps:
- the epidermal model according to the invention can also be used in any method, especially automated, for screening or identifying new cosmetic, dermatological or pharmaceutical compounds.
- Screening methods for identifying novel effective compounds generally include contacting said test compound with a model of skin and / or epidermis obtained according to the invention and then a step of reading the effect of the compound on said model, in particular by comparing this effect with a control or control model according to the invention that has not been contacted with the test compound.
- This last reading step may also be performed by determining or analyzing epidermal markers and / or associated cells and included in the solidified matrix, such as proteins associated with these structures. For example, if these associated cells are cells of the immune system, said screening method will for example be intended to identify or select test compounds capable of inducing undesired side effects such as allergic reactions.
- the products tested may also be gene expression vectors, or nucleic acids, such as antisense nucleic acids, microR A, siR A capable of modifying the expression of a constitutive gene of cells present in said model of expression. the invention.
- the present invention relates to a method for determining the therapeutic treatment adapted for an individual suffering from a disorder of the skin, in particular of the epidermis, this method comprising the following steps:
- the present invention relates to a method for determining the therapeutic treatment adapted for an individual suffering from a disorder of the skin, in particular of the epidermis, according to the present invention, characterized in that at the step a), the skin fragment used is a fragment from a collection or skin culture.
- the present invention relates to the use of a model according to the present invention, for determining for a product the side effects on the epidermis, such as for example:
- Such a model may indeed be used according to the present invention to be used or implemented in any method or test in vitro or ex vivo requiring animal or human experiments, such as, for example, the release or penetration study of active principles, and or of their cutaneous bioavailability, the study of their effectiveness, or even of their tolerance, of their compatibility, these active principles being for cosmetic, dermatological and pharmaceutical purposes.
- said product is a cosmetic, dermatological or pharmaceutical product.
- the use as a model will also concern its implementation for the study of any pathology resulting in abnormalities of the skin, in particular of the epidermis, but also for all the cells located under the epidermis for which the epidermis constitutes a natural barrier and for which topical therapy is feasible.
- Step 1 A cylindrical skin biopsy is performed
- Step 2 The biopsy is placed in a container containing an enzymatic solution degrading the basement membrane;
- Step 3 The biopsy is incubated at 4 ° C until the dermis detaches from the epidermis;
- Step 4 The epidermis is placed on a solution of blood plasma
- Step 5 The blood plasma solution is solidified by coagulation.
- Step 6 The epidermis disposed on the coagulated plasma is maintained in culture.
- EXAMPLE Different steps of the process (see figure) 1. A cylindrical biopsy is first performed on a fragment of skin freshly removed to allow its survival ex vivo.
- the skin biopsy is gently deposited in an enzyme solution of dispase, trypsin or thermolysin, at about 10 mg / ml of solution which specifically degrades the basement membrane proteins, so that the superficial part (the epidermis) remains emerging while the underlying dermis is submerged.
- the whole is allowed to incubate (usually at least 10 hours at 4 ° C) until the dermis gradually sinks into the solution after being detached from the epidermis remained, when to him, spread , floating on the surface of the solution, and always associated with its annexes.
- the separation between the epidermis and the dermis can be completed using forceps while ensuring the integrity of the tissues.
- the enzymatic solution is then replaced by a solution derived from blood plasma treated with an anti-coagulant agent with reversible properties in the presence of calcium ions (sodium citrate).
- This solution contains 42% blood plasma, 50% 0.9% NaCl solution, 8% 1% CaCl 2 saline and an anti-fibrinolytic agent (tranaxemic acid or aprotinin). 5.
- the plasma acts as a dermal support on which adheres the epidermis explant.
- Coagulation consists essentially of the transformation, in the presence of calcium ions and thrombin, of the fibrinogen present in the plasma into a scaffold of fibrin molecules linked together by covalent bonds.
- the anti-fibrinolytic agent functions to inhibit the enzymes capable of degrading the plasma matrix secreted by the epidermis explant, and thus to maintain the integrity of the dermal support.
- the plasma solution may be substituted with a solution of fibrinogen or collagen. For the latter, incubation at 37 ° C alone allows solidification.
- the complex consisting of the explant of epidermis is kept in culture with the epidemic in contact with the air.
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Abstract
Description
PROCÈDE IN VITRO OU EX VIVO POUR LA CONSERVATION ET/OU LA MAINTENANCE EN PROCESS IN VITRO OR EX VIVO FOR CONSERVATION AND / OR MAINTENANCE IN
SURVIE D'UN EPIDERME. SURVIVAL OF AN EPIDERM.
La présente invention a pour objet sur un procédé in vitro ou ex vivo pour la conservation et/ou la maintenance en survie d'un épiderme. The present invention relates to an in vitro or ex vivo process for the preservation and / or survival maintenance of an epidermis.
Sous un autre aspect, la présente invention comprend le modèle d'épiderme ainsi obtenu et sur son utilisation dans un kit ou dans procédé pour le criblage ou la sélection de composés cosmétiques ou pharmaceutiques. In another aspect, the present invention includes the epidermal model thus obtained and its use in a kit or method for screening or selecting cosmetic or pharmaceutical compounds.
L' épiderme est un épithélium pavimenteux pluristratifié et squameux. Il forme une barrière qui résiste aux ravages de la dessiccation, ainsi qu' aux agressions mécaniques, chimiques et microbiennes. Le type cellulaire principal constituant l'épidémie est le kératinocyte. Ce tissu comprend également d'autres populations cellulaires telles que les mélanocytes, les cellules de Langerhans, et les cellules de Merkel. L'épiderme est conventionnellement sous divisé en quatre strates distinctes comprenant des couches internes vers les couches les plus superficielles : la couche basale (une couche), la couche épineuse (4-15 couches), la couche granuleuse (1-3 couches) et la couche cornée (5-10 couches). L'épiderme repose sur le derme grâce une membrane basale constituée entre autres de molécules de collagène. Le derme contient des réseaux vasculaires et nerveux très denses ainsi que les annexes épidermiques, structures kératinisées prolongeant l'épiderme et incluant les follicules pileux, les glandes sébacées, et les glandes sudoripares. The epidermis is a squamous and squamous squamous epithelium. It forms a barrier that resists the ravages of desiccation, as well as mechanical, chemical and microbial attacks. The main cell type constituting the epidemic is the keratinocyte. This tissue also includes other cell populations such as melanocytes, Langerhans cells, and Merkel cells. The epidermis is conventionally subdivided into four distinct layers comprising inner layers towards the most superficial layers: the basal layer (one layer), the spinous layer (4-15 layers), the granular layer (1-3 layers) and the stratum corneum (5-10 layers). The epidermis rests on the dermis thanks to a basement membrane composed among others of collagen molecules. The dermis contains very dense vascular and nervous networks as well as epidermal appendages, keratinized structures extending the epidermis and including hair follicles, sebaceous glands, and sweat glands.
Les techniques qui permettent d'obtenir des épidermes reconstruits in vitro sont dérivées de l'ingénierie cellulaire. Les découvertes réalisées par Rheinwald et Green (Rheinwald, J. G. et al, "Sériai cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells", Cell, 6 (1975), 331-344; Green, H. et al., "Growth of cultured human epidermal cells into multiple epithelia suitable for grafting", Proc. Nat. Acad. Sci. USA 76 (1979), 5665-5668) ont été le départ de l'établissement de protocoles de cultures de kératinocytes humains, et de leurs utilisations en médecine (grand brûlés) ainsi qu'en recherche dermatologique (modèles in vitro). De nombreux protocoles permettant le développement d'épidermes/peaux reconstruits ont été établis. On peut citer par exemple ceux qui sont décrits dans les brevets ou dans les demandes de brevets EP 285471, EP 285474, EP 418035, WO- A-90 02796, WO-A-9116010, EP 197090, EP 20753, FR 2665175, FR 2689904. Techniques for obtaining reconstructed epidermis in vitro are derived from cellular engineering. The findings by Rheinwald and Green (Rheinwald, JG et al, "Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells", Cell, 6 (1975), 331-344; al., "Growth of cultured human epidermal cells into multiple epithelia suitable for grafting", Proc Nat Sci USA 76 (1979), 5665-5668) were the starting point for the establishment of keratinocyte culture protocols. humans, and their uses in medicine (burns) and in dermatological research (in vitro models). Many protocols for the development of reconstructed epidermis / skin have been established. Examples which may be mentioned are those described in the patents or in the patent applications EP 285471, EP 285474, EP 418035, WO-A-90 02796, WO-A-9116010, EP 197090, EP 20753, FR 2665175, FR. 2689904.
De manière très générale, les modèles de peau ou d'épidermes reconstruits décrits dans ces documents sont constitués de kératinocytes humains déposés sur un support, souvent un équivalent de derme, et cultivés dans des conditions telles qu'ils entrent dans un programme de différenciation aboutissant à la formation d'un équivalent d'épiderme. In a very general manner, the reconstructed skin or epidermis models described in these documents consist of human keratinocytes deposited on a support, often a dermis equivalent, and cultured under conditions such that they enter into a differentiation program resulting in to the formation of an epidermis equivalent.
Les modèles d'épidermes ou de peau reconstruits sont fréquemment utilisés pour des investigations en dermatologie dans le cadre des tests de substances, comme pour les candidats médicaments ou les actifs cosmétiques en développement. Les effets pharmacologiques de ces molécules, notamment leur potentiel corrosif ou l'irritant, sont également analysés grâce à ces systèmes. Les épidermes reconstruits sont aussi utiles pour répondre à des questions de type moléculaires, immuno logiques ou encore histologiques. Ceci comprend les études de pénétration et d'absorption. En comparaison avec les études précliniques utilisant des animaux et les études cliniques chez l'Homme, les épidermes/peaux reconstruits permettent d'obtenir des résultats plus rapidement et de façon plus reproductible. Reconstructed epidermal or skin models are frequently used for dermatology investigations in substance testing, as for drug candidates or developing cosmetic actives. The pharmacological effects of these molecules, in particular their corrosive potential or the irritant, are also analyzed thanks to these systems. The reconstructed epidermis are also useful for answering questions of the molecular, immunological or histological type. This includes penetration and absorption studies. In comparison with preclinical studies using animals and clinical studies in humans, reconstructed epidermis / skins allow results to be obtained more quickly and in a more reproducible way.
Afin d'obtenir un épiderme comportant plusieurs types cellulaires, des mélanocytes ou des cellules immunitaires peuvent être ajoutées aux cultures de kératinocytes (Céline AUXENFANS et al, « Evolution of three dimensional skin équivalent models reconstructed in vitro by tissue engineering », Eur J Dermatol 2009; 19 (2): 107-13). Cependant, les technologies utilisées ne permettent pas encore une production d'épidermes contenant à la fois des kératinocytes, des mélanocytes, des cellules de Langerhans et des cellules de Merkel. Enfin, de nombreux verrous technologiques persistent pour reconstruire in vitro des annexes épidermiques comme des follicules pileux, des glandes sébacées et des glandes sudoripares. En conclusion, les technologies de reconstruction disponibles jusqu'alors sont encore très limitantes pour permettre la production d'un épiderme contenant l'ensemble des annexes et types cellulaires qui le caractérise. Ainsi, depuis de nombreuses années, la mise au point de nouveau procédés visant à obtenir des modèles de peau reconstruite est recherchée. De tels modèles auraient comme avantages de pouvoir permettre la réalisation d'études nécessaires à une meilleure compréhension du rôle de la peau, notamment de son épiderme tant dans le domaine mécanique que dans le domaine physiologique. De tels modèles peuvent également être utilisés pour la prédiction par des tests in vitro ou ex vivo, de l'activité de principe actifs cosmétiques et/ou pharmaceutiques ou encore des effets secondaires de composé topiques. In order to obtain an epidermis comprising several cell types, melanocytes or immune cells can be added to the keratinocyte cultures (Céline AUXENFANS et al, "Evolution of three dimensional skin equivalents models reconstructed in vitro by tissue engineering", Eur J Dermatol 2009 19 (2): 107-13). However, the technologies used do not yet allow the production of epidermis containing both keratinocytes, melanocytes, Langerhans cells and Merkel cells. Finally, many technological barriers persist to reconstruct in vitro epidermal appendages such as hair follicles, sebaceous glands and sweat glands. In conclusion, the reconstruction technologies available until now are still very limiting to allow the production of an epidermis containing all the annexes and cell types that characterizes it. Thus, for many years, the development of new methods for obtaining models of reconstructed skin is sought. Such models would have the advantages of being able to allow the realization of studies necessary for a better understanding of the role of the skin, in particular of its epidermis both in the mechanical field and in the physiological field. Such models can also be used for the prediction by in vitro or ex vivo tests, of the activity of cosmetic and / or pharmaceutical active ingredients or else of the topical compound side effects.
Ceci est justement l'objet de la présente invention. This is precisely the object of the present invention.
De façon surprenante, la demanderesse a mis en évidence que la mise en œuvre du procédé de l'invention tel que décrit ci-après, notamment comprenant le dépôt de fragment de peau dans une première étape sur une phase liquide, puis, dans une seconde étape, sur une phase liquide dont on peut induire la solidification, permettait d'obtenir la conservation ou la maintenance en survie d'un explant d'épiderme pouvant être utilisable comme modèle in vitro ou ex vivo. Surprisingly, the applicant has demonstrated that the implementation of the method of the invention as described below, in particular comprising the deposition of skin fragment in a first step on a liquid phase, then, in a second stage, on a liquid phase which can induce solidification, allowed to obtain preservation or survival maintenance of an explant epidermis that can be used as an in vitro model or ex vivo.
La présente invention a donc pour objet un procédé in vitro ou ex vivo pour la conservation ou la maintenance en survie d'un explant épiderme. The subject of the present invention is therefore an in vitro or ex vivo method for the preservation or survival maintenance of an explant epidermis.
L'invention comprend en particulier un procédé permettant de faire survivre ex vivo un explant entier d'épiderme avec ces annexes. The invention particularly includes a method for ex vivo survival of an entire epidermis explant with these appendices.
De préférence, ledit fragment de peau est un fragment de peau de mammifère, de préférence humaine. Preferably, said skin fragment is a fragment of mammalian skin, preferably human skin.
La présente invention se rapporte également à un épiderme, de préférence isolé, susceptible d'être obtenu ou directement obtenu par un tel procédé et son utilisation comme modèle pour évaluer ex vivo ou in vitro l'ef et de produits cosmétique, pharmaceutique ou dermatologique à administration topique. The present invention also relates to an epidermis, preferably isolated, obtainable or directly obtained by such a method and its use as a model for evaluating ex vivo or in vitro ef and cosmetic, pharmaceutical or dermatological products to topical administration.
Ainsi, sous un premier aspect, la présente invention a pour objet un procédé ex vivo ou in vitro de conservation ou de maintenance en survie d'un épiderme à partir d'un fragment de peau comprenant de l'épiderme et du derme, ce fragment ayant été préalablement prélevé chez un mammifère, caractérisé en ce que ledit procédé comprend les étapes suivantes : Thus, in a first aspect, the present invention relates to an ex vivo or in vitro method of preservation or maintenance in survival of an epidermis from a skin fragment comprising epidermis and dermis, this fragment having been previously taken from a mammal, characterized in that said method comprises the following steps:
a) la préparation d'une solution enzymatique dans un container, ladite solution enzymatique comprenant au moins une protéase ; b) le dépôt dudit fragment sur ladite solution enzymatique de façon à ce que l'épiderme contenu dans ledit fragment de peau reste émergé avec sa surface sèche et au contact de l'air ambiant, et le derme sous-jacent dudit fragment soit immergé dans ladite solution enzymatique ; a) the preparation of an enzymatic solution in a container, said enzymatic solution comprising at least one protease; b) depositing said fragment on said enzymatic solution so that the epidermis contained in said skin fragment remains emerged with its dry surface and in contact with the ambient air, and the underlying dermis of said fragment is immersed in said enzymatic solution;
c) l'incubation dudit fragment de peau ainsi disposé dans la solution enzymatique pendant une période suffisante pour aboutir au détachement du derme de l'épiderme; c) incubating said skin fragment thus disposed in the enzymatic solution for a period sufficient to result in detachment of the dermis from the epidermis;
d) le cas échéant, l'élimination du derme ainsi détaché ; (d) where appropriate, the removal of the detached dermis;
e) le remplacement de la solution enzymatique par une matrice liquide, de préférence nutritive, apte à pouvoir se solidifier sous l'action d'une augmentation de la température ou d'un composé ou composition spécifique ; e) replacing the enzymatic solution with a liquid matrix, preferably nutritive, able to be solidified under the action of an increase in temperature or a specific compound or composition;
f) la solidification de la matrice augmentation de la température de ladite matrice et/ou par l'ajout dudit composé ou composition spécifique. f) solidifying the matrix, increasing the temperature of said matrix and / or adding said specific compound or composition.
Ce procédé se révèle avantageux par le fait qu'il permet d'obtenir un modèle d'épiderme qui peut être conservé ou maintenu en survie de manière prolongée sans dégradation notable. This method is advantageous in that it makes it possible to obtain an epidermis model that can be preserved or maintained in prolonged survival without appreciable degradation.
De préférence, ledit épiderme comprend l'ensemble de ses couches cellulaires ainsi que les annexes épidermiques dans leur totalité. Preferably, said epidermis comprises all of its cell layers as well as the epidermal appendices in their entirety.
Par « ensemble de ses couches cellulaires » pour un épiderme, on entend désigner ici la couche basale, la couche épineuse, la couche granuleuse et la couche cornée. By "set of its cell layers" for an epidermis is meant here the basal layer, the spinous layer, the granular layer and the stratum corneum.
Par « annexes épidermiques dans leur totalité», on entend désigner ici les follicules pileux, les glandes sébacées et les glandes sudoripares. By "epidermal appendices in their entirety" is meant here hair follicles, sebaceous glands and sweat glands.
De préférence, ladite au moins protéase contenue dans la solution enzymatique est une protéase dont l'activité permet de séparer l'épiderme du derme, notamment par dégradation des protéines de la membrane basale séparant l'épiderme du derme. Preferably, said at least one protease contained in the enzyme solution is a protease whose activity makes it possible to separate the epidermis from the dermis, in particular by degradation of the basement membrane proteins separating the epidermis from the dermis.
De préférence, ladite protéase est choisie parmi la famille des dispases, trypsines ou thermolysines. Preferably, said protease is chosen from the family of dispases, trypsins or thermolysins.
De préférence, à l'étape a) du procédé de l'invention, le niveau de ladite solution enzymatique dans le container (profondeur) est supérieur à l'épaisseur du fragment prélevé. De préférence la solution enzymatique est une solution aqueuse, de préférence physiologique, de préférence compatible avec la conservation et /ou la maintenance en survie ex vivo ou in vitro de l'épiderme, tel que par exemple un tampon phosphate salin (PBS), un tampon HBSS (Hank's balanced sait solution) ou tout autre tampon ou solution physiologique, tel que une solution aqueuse NaCl 0, 9 % (V/V), compatible avec la conservation et/ou la maintenance en survie et/ou la culture de cellules de mammifère. Preferably, in step a) of the process of the invention, the level of said enzymatic solution in the container (depth) is greater than the thickness of the sampled fragment. Preferably the enzymatic solution is an aqueous solution, preferably physiological, preferably compatible with the preservation and / or maintenance in ex vivo or in vitro survival of the epidermis, such as for example a phosphate buffered saline (PBS), a Hank's balanced solution (HBSS) or any other buffer or physiological solution, such as a 0.9% aqueous NaCl solution (V / V), compatible with the preservation and / or maintenance in survival and / or cell culture of mammal.
Par culture de cellules, on entend désigner ici en particulier la maintenance de l'état physiologique de la cellule. By cell culture is meant here in particular the maintenance of the physiological state of the cell.
De préférence, à l'étape c) du procédé de l'invention, ledit détachement est induit par les protéases contenues dans la solution enzymatique. Preferably, in step c) of the process of the invention, said detachment is induced by the proteases contained in the enzymatic solution.
De préférence, à l'étape c) du procédé de l'invention, le derme ainsi détaché plonge progressivement dans le fond du container. Preferably, in step c) of the process of the invention, the dermis thus detached progressively dips into the bottom of the container.
L'invention comprend en particulier un procédé selon l'invention permettant de conserver et/ou de faire survivre ex vivo ou in vitro un explant entier d' épidémie avec ces annexes, ledit procédé comprenant les étapes suivantes : The invention particularly comprises a method according to the invention for preserving and / or surviving ex vivo or in vitro an entire explant of epidemic with these appendices, said method comprising the following steps:
(i) la disposition d'une biopsie de peau cylindrique dans une solution enzymatique de façon à ce que la ligne de flottaison sépare l'épiderme émergé du derme immergé; (i) disposing a cylindrical skin biopsy in an enzymatic solution so that the water line separates the emerged epidermis from the submerged dermis;
(ii) la dégradation enzymatique de la membrane basale de telle manière que l'épiderme flotte à la surface tandis que le derme se détache et plonge progressivement dans le fond; (ii) enzymatic degradation of the basement membrane so that the epidermis floats on the surface while the dermis detaches and progressively dives into the bottom;
(iii) la disposition de l'épiderme sur une matrice liquide, telle qu'une solution de plasma sanguin, solution de fïbrinogène ou de collagène; et (iii) the provision of the epidermis on a liquid matrix, such as a solution of blood plasma, fibrinogen solution or collagen; and
(iv) la solidification de ladite matrice par ajout de thrombine ou par la chaleur. (iv) solidifying said matrix by adding thrombin or by heat.
L'invention porte sur l'utilisation un tel épiderme obtenu par un procédé selon la présente invention à des fins de modèle ex vivo ou in vivo, particulier pour des analyses toxico logiques ou à des fins de recherche. De préférence, lesdites analyses toxicologiques sont choisies parmi les tests de sensibilisation, d'absorption, de métabolisme, de corrosion, ou d'irritation se la peau, de préférence de l'épiderme. The invention relates to the use of such an epidermis obtained by a method according to the present invention for model ex vivo or in vivo, particularly for toxicological analyzes or for research purposes. Preferably, said toxicological analyzes are chosen from sensitization, absorption, metabolism, corrosion, or irritation tests on the skin, preferably the epidermis.
Selon un mode de réalisation préféré, ledit procédé comprend à l'étape e): i) le prélèvement de l'épiderme reposant sur la solution enzymatique et son dépôt sur ladite matrice liquide apte à pouvoir se solidifier, cette matrice liquide étant contenue dans un second container; ou According to a preferred embodiment, said method comprises in step e): i) the removal of the epidermis resting on the enzymatic solution and its deposition on said liquid matrix capable of being able to solidify, this liquid matrix being contained in a second container; or
ii) le remplacement de la solution enzymatique par une matrice liquide dans le container initial. ii) replacing the enzymatic solution with a liquid matrix in the initial container.
Selon un mode de réalisation également préféré, ledit procédé comprend à l'étape e): According to a also preferred embodiment, said method comprises in step e):
i) le prélèvement de l'épiderme reposant sur la solution enzymatique et son dépôt sur ladite matrice liquide apte à pouvoir se solidifier contenue dans un second container; et, i) the removal of the epidermis based on the enzymatic solution and its deposit on said liquid matrix capable of being solidified contained in a second container; and,
en ce que ladite matrice liquide apte à pouvoir se solidifier contient en outre des cellules autres que des cellules de l'épiderme, de préférence choisies dans le groupe de cellules constitué par les fibroblastes, des cellules endothéliales, des cellules nerveuses, des cellules constituant le derme et l'hypoderme et des cellules immunitaires ; ou in that said solidifying liquid matrix further contains cells other than cells of the epidermis, preferably selected from the group of cells consisting of fibroblasts, endothelial cells, nerve cells, cells constituting the dermis and hypodermis and immune cells; or
ii) le remplacement de la matrice contenant la solution enzymatique par une matrice liquide dans le container initial, ladite matrice liquide de remplacement étant une matrice liquide apte à pouvoir se solidifier, et, le cas échéant contenant en outre des cellules autres que des cellules de l'épiderme, de préférence choisies dans le groupe de cellules constitué par les fibroblastes, des cellules endothéliales, des cellules nerveuses, des cellules constituant le derme et l'hypoderme et des cellules immunitaires. ii) replacing the matrix containing the enzymatic solution with a liquid matrix in the initial container, said replacement liquid matrix being a liquid matrix capable of being able to solidify, and, where appropriate, further containing cells other than epidermis, preferably selected from the group of cells consisting of fibroblasts, endothelial cells, nerve cells, cells constituting the dermis and hypoderm and immune cells.
Selon un autre mode de réalisation également préféré, à l'étape e) dudit procédé, le fragment obtenu est ensuite placé sur une matrice liquide apte à pouvoir se solidifier ladite matrice liquide pouvant apporter tous les ingrédients nutritifs et/ou nécessaires à sa culture, en particulier à la maintenance de l'état de physiologie initiale des cellules qui le constituent. Selon un autre mode de réalisation également préféré, à l'étape e) dudit procédé, le fragment obtenu est ensuite placé sur une matrice liquide apte à pouvoir se solidifier ladite matrice liquide pouvant apporter en outre tous les ingrédients et toutes les cellules nécessaires à la préparation d'un équivalent de peau complet reproduisant les caractéristiques d'une peau in vivo. According to another also preferred embodiment, in step e) of said process, the fragment obtained is then placed on a liquid matrix capable of being able to solidify said liquid matrix capable of supplying all the nutritive and / or necessary ingredients for its cultivation, in particular to the maintenance of the state of initial physiology of the cells which constitute it. According to another also preferred embodiment, in step e) of said method, the fragment obtained is then placed on a liquid matrix capable of being able to solidify said liquid matrix that can additionally provide all the ingredients and all the cells necessary for the preparation of a complete skin equivalent reproducing the characteristics of a skin in vivo.
De préférence, ladite matrice liquide ne recouvre pas la face supérieure de l'épiderme avant que cette matrice ne soit solidifiée. Preferably, said liquid matrix does not cover the upper face of the epidermis before this matrix is solidified.
Sous un deuxième aspect, la présente invention a pour objet un procédé ex vivo ou in vitro de conservation ou de maintenance en survie d'un épiderme à partir d'un fragment de peau, ledit procédé comprend les étapes suivantes : In a second aspect, the present invention relates to an ex vivo or in vitro method of preservation or maintenance in survival of an epidermis from a skin fragment, said method comprises the following steps:
A) la conservation ou maintenance en survie d'un épiderme à partir d'un fragment de peau préalablement prélevé par un procédé selon la présente invention; et A) the preservation or survival maintenance of an epidermis from a skin fragment previously removed by a method according to the present invention; and
B) la culture de l'épiderme ainsi obtenue à l'étape A) dans des conditions de cultures adéquates, de préférence, ledit épiderme étant au contact de l'air. B) the culture of the epidermis thus obtained in step A) under suitable culture conditions, preferably, said epidermis being in contact with the air.
Plus spécifiquement, le procédé selon l'invention est caractérisé en ce que ledit épiderme est un épiderme entier avec ses annexes, comprenant les follicules pileux, les glandes sébacées et sudoripares. More specifically, the process according to the invention is characterized in that said epidermis is an entire epidermis with its appendages, comprising the hair follicles, the sebaceous and sweat glands.
Selon un mode de réalisation également préféré, ledit fragment préalablement prélevé est un fragment de forme cylindrique, de préférence dont la taille est comprise entre 1 à 10 mm de diamètre, de préférence encore de 1 à 8 mm et de 0,5 à 8 mm d'épaisseur, de préférence de 1 à 5 mm. According to a also preferred embodiment, said fragment previously removed is a fragment of cylindrical shape, preferably whose size is between 1 to 10 mm in diameter, more preferably from 1 to 8 mm and from 0.5 to 8 mm thickness, preferably 1 to 5 mm.
Selon un mode de réalisation également préféré, la solution enzymatique apte à dégrader les protéines de la membrane basale séparant l'épiderme du derme et contenue dans la ladite matrice à l'étape a) est une solution contenant une protéase, de préférence de la dispase, de la trypsine ou de la thermo lysine, de préférence à une concentration comprise entre 1 et 20 mg/ml (V/V) de solution, de préférence encore entre 5 et 15 mg/ml et entre 7, 5 et 12, 5 mg/ml de solution. According to a also preferred embodiment, the enzymatic solution able to degrade the proteins of the basement membrane separating the epidermis from the dermis and contained in the said matrix in step a) is a solution containing a protease, preferably dispase. , trypsin or thermolysin, preferably at a concentration of between 1 and 20 mg / ml (V / V) of solution, more preferably between 5 and 15 mg / ml and between 7.5 and 12.5. mg / ml solution.
Selon un mode de réalisation également préféré du procédé selon l'invention, à l'étape e), ladite matrice liquide apte à pouvoir se solidifier sous l'action d'une augmentation de la température ou d'un composé ou composition spécifique est choisie parmi toute solution liquide, de préférence nutritive, capable de se solidifier ou de se gélifier dans des conditions particulières compatibles avec la survie et la culture de cellules d' épidémie, de préférence choisie parmi le plasma sanguin ou une solution dérivée de plasma sanguin, notamment dilué en tampon physiologique à au maximum 10 %, de préférence à au moins 20 %, au moins 30 % et 40 %, une solution de fïbrinogène, une solution de collagène, de la gélatine, les gels polymériques synthétiques, naturels tels que les gels d'agarose, d'amidon ou de polysaccharides. According to a also preferred embodiment of the process according to the invention, in stage e), said liquid matrix capable of being able to solidify under the action of an increase in temperature or of a specific compound or composition is chosen from any liquid solution, preferably nutritive, capable of solidifying or gelling under particular conditions compatible with the survival and culture of epidemic cells, preferably chosen from blood plasma or a solution derived from blood plasma, in particular diluted in physiological buffer to at most 10%, preferably at least 20%, at least 30% and 40%, a fibrinogen solution, a collagen solution, gelatin, natural synthetic polymeric gels such as agarose, starch or polysaccharide gels.
Selon un mode de réalisation également préféré du procédé selon l'invention, à l'étape e) : According to a also preferred embodiment of the method according to the invention, in step e):
- ladite matrice liquide apte à pouvoir se solidifier est une solution liquide dérivée de plasma sanguin traité avec un agent anticoagulant aux propriétés réversibles, de préférence par du citrate de sodium ; et said liquid matrix capable of being able to solidify is a liquid solution derived from blood plasma treated with an anticoagulant agent with reversible properties, preferably with sodium citrate; and
- la solidification de ladite matrice est obtenue en présence d'ions calcium, de préférence également en présence de thrombine. the solidification of said matrix is obtained in the presence of calcium ions, preferably also in the presence of thrombin.
Selon un mode de réalisation également préféré du procédé selon l'invention, à l'étape c), ledit fragment de peau ainsi disposé sur la solution est incubé pendant une période d'au moins 10 heures, de préférence à 4°C, de préférence encore une nuit à 4 °C. According to a also preferred embodiment of the method according to the invention, in step c), said skin fragment thus disposed on the solution is incubated for a period of at least 10 hours, preferably at 4 ° C., of more preferably overnight at 4 ° C.
Selon un mode de réalisation également préféré du procédé selon l'invention, à l'étape e), ladite matrice liquide est solidifiée après au maximum de 8 heures, de préférence moins de 2 heures ou moins d'une heure, une durée de moins de 30 min, de préférence de moins de 10 min étant la durée la plus préférée pour engager la phase de solidification de la matrice liquide. According to a also preferred embodiment of the method according to the invention, in step e), said liquid matrix is solidified after a maximum of 8 hours, preferably less than 2 hours or less than one hour, a duration of less than 30 min, preferably less than 10 min being the most preferred time to initiate the solidification phase of the liquid matrix.
Selon un mode de réalisation également préféré du procédé selon l'invention, à l'étape e), ladite matrice liquide apte à pouvoir se solidifier est une solution dérivée de plasma sanguin contenant de 25 % à 60 %, de préférence entre According to a also preferred embodiment of the method according to the invention, in step e), said liquid matrix capable of being able to solidify is a solution derived from blood plasma containing from 25% to 60%, preferably between
35 % et 45 % (v/v) de plasma sanguin, de 70 % à 35 % d'une solution physiologique, telle qu'une solution de NaCl à 0,9 %, de 5 % à 12 %, de préférence 8 %, d'une solution saline de CaCl2 à 1 % et d'un agent anti- fîbrinolytique en concentration suffisante pour obtenir l'activité anti-fribrinolytique recherchée, de préférence entre 5 % et 2 %, de préférence l'agent anti-fibrinolytique étant choisi parmi l'acide tranaxémique ou de l'aprotinine. Selon un mode de réalisation également préféré du procédé selon l'invention, à l'étape e), ladite matrice liquide apte à pouvoir se solidifier est une solution liquide de fïbrinogène et de thrombine, ou de collagène et pour laquelle l'incubation à 37 °C permet sa solidification. 35% and 45% (v / v) of blood plasma, 70% to 35% of a physiological solution, such as a solution of NaCl 0.9%, 5% to 12%, preferably 8% , a 1% CaCl 2 saline solution and an anti-fibrinolytic agent in sufficient concentration to obtain the desired anti-fibrinolytic activity, preferably between 5% and 2%, preferably the anti-fibrinolytic agent. being selected from tranaxemic acid or aprotinin. According to a also preferred embodiment of the process according to the invention, in stage e), said solidifying liquid matrix is a liquid solution of fibrinogen and thrombin, or collagen and for which the incubation at 37.degree. ° C allows its solidification.
Sous un troisième aspect, la présente invention a pour objet un modèle d'épiderme susceptible d'être obtenu ou directement obtenu par le procédé selon la présente invention. In a third aspect, the present invention relates to an epidermal model that can be obtained or directly obtained by the method according to the present invention.
Selon cet aspect de l'invention, a également pour objet un kit comprenant un tel modèle d'épiderme reposant notamment sur une matrice solide et, le cas échéant, nutritive, cette matrice solide étant adaptée par sa composition à la conservation et/ou la maintenance en survie dudit épiderme, le cas échéant à multiplication et à la différenciation des cellules pouvant être incluses dans cette matrice solide. According to this aspect of the invention, a further object is also a kit comprising such an epidermis model resting in particular on a solid and, where appropriate, nutritive matrix, this solid matrix being adapted by its composition to the preservation and / or the maintenance in survival of said epidermis, where appropriate to the multiplication and differentiation of the cells that may be included in this solid matrix.
Ainsi, selon cet aspect, l'invention a pour objet également un kit d'évaluation de composé cosmétique, dermatologique ou thérapeutique pour la peau comprenant un modèle de peau ou d'épiderme obtenu selon le procédé de l'invention. Thus, according to this aspect, the subject of the invention is also a kit for evaluation of a cosmetic, dermatological or therapeutic compound for the skin comprising a skin or epidermis model obtained according to the method of the invention.
Un autre objet de la présente invention se rapporte à une méthode de screening in vitro de composés candidats pour le traitement cosmétique ou thérapeutique de la peau, de préférence de Γ épiderme, de préférence de Γ épiderme associé au derme comprenant les étapes suivantes: Another subject of the present invention relates to a method for in vitro screening of candidate compounds for the cosmetic or therapeutic treatment of the skin, preferably of the epidermis, preferably of the epidermis associated with the dermis, comprising the following steps:
a) l'obtention d'un modèle d'épiderme selon la présente invention; a) obtaining an epidermis model according to the present invention;
b) la mise en contact dudit modèle avec le composé candidat ; et b) contacting said model with the candidate compound; and
c) la mise en évidence d'une modification physiologique, en particulier la perméabilité, ou morphogénique des cellules de l'épiderme dudit modèle; et d) la sélection dudit composé si les modifications obtenues sont celles souhaitées pour le traitement. c) the demonstration of a physiological modification, in particular the permeability or morphogenicity of the cells of the epidermis of said model; and d) selecting said compound if the modifications obtained are those desired for the treatment.
Ainsi le modèle d'épiderme selon l'invention peut aussi être utilisé dans tout procédé, notamment automatisé, pour le criblage ou l'identification de nouveaux composés cosmétiques, dermatologiques ou pharmaceutiques. Thus, the epidermal model according to the invention can also be used in any method, especially automated, for screening or identifying new cosmetic, dermatological or pharmaceutical compounds.
Les procédés de criblage en vue d'identifier de nouveaux composés efficace comprennent en général une de mise en contact dudit composé à tester avec un modèle de peau et/ou d'épiderme obtenu selon l'invention puis une étape de lecture de l'effet du composé sur ledit modèle, notamment en comparant cet effet avec un modèle contrôle ou témoin selon l'invention n'ayant pas été mis en contact avec le composé à tester. Cette dernière étape de lecture pourra également être réalisée par la détermination ou l'analyse de marqueurs épidermiques et/ou de cellules associées et incluses dans la matrice solidifiée, tels que des protéines associées à ces structures. Par exemple si ces cellules associées sont des cellules du système immunitaire, ledit procédé de criblage sera par exemple destiné à identifier ou sélectionner des composés tests susceptibles d'induire des effets secondaires non recherchés tels que des réactions allergiques. Screening methods for identifying novel effective compounds generally include contacting said test compound with a model of skin and / or epidermis obtained according to the invention and then a step of reading the effect of the compound on said model, in particular by comparing this effect with a control or control model according to the invention that has not been contacted with the test compound. This last reading step may also be performed by determining or analyzing epidermal markers and / or associated cells and included in the solidified matrix, such as proteins associated with these structures. For example, if these associated cells are cells of the immune system, said screening method will for example be intended to identify or select test compounds capable of inducing undesired side effects such as allergic reactions.
Les produits testés pourront également être des vecteurs d'expression de gènes, ou encore des acides nucléiques, tels que des acides nucléiques antisens, microR A, siR A aptes à modifier l'expression d'un gène constitutif de cellules présentes dans ledit modèle de l'invention. The products tested may also be gene expression vectors, or nucleic acids, such as antisense nucleic acids, microR A, siR A capable of modifying the expression of a constitutive gene of cells present in said model of expression. the invention.
D'autres effets tels que l'expression de certains médiateurs ou la cytotoxicité intrinsèque des composés tests vis à vis de certaines cellules pourront être également recherchés comme effet secondaire selon les cellules associées également présentes dans la matrice solidifiée. Other effects such as the expression of certain mediators or the intrinsic cytotoxicity of the test compounds with respect to certain cells may also be sought as a side effect according to the associated cells also present in the solidified matrix.
Ces modèles témoins seront bien entendus mis en œuvre dans les mêmes conditions que le modèle de l'invention ayant reçu le produit à tester. These control models will of course be used under the same conditions as the model of the invention which has received the product to be tested.
Sous un cinquième aspect, la présente invention a pour objet un procédé pour déterminer le traitement thérapeutique adapté pour un individu souffrant d'un désordre de la peau, notamment de l'épiderme, ce procédé comprenant les étapes suivantes : In a fifth aspect, the present invention relates to a method for determining the therapeutic treatment adapted for an individual suffering from a disorder of the skin, in particular of the epidermis, this method comprising the following steps:
a) à partir d'un fragment de peau prélevé chez ledit patient, l'obtention d'un modèle selon la présente invention; a) from a skin fragment taken from said patient, obtaining a model according to the present invention;
b) la mise en contact dudit modèle avec un composé candidat pour ledit traitement ; c) la mise en évidence de modification physiologique ou morphogénique associée à l'efficacité du traitement des cellules de l'épiderme pour ledit désordre de l'épiderme; et b) bringing said model into contact with a candidate compound for said treatment; c) the demonstration of physiological or morphogenic modification associated with the effectiveness of the treatment of the cells of the epidermis for said epidermal disorder; and
d) la sélection dudit composé si les modifications obtenues sont celles souhaitées pour le traitement. Sous un sixième aspect, la présente invention a pour objet un procédé pour déterminer le traitement thérapeutique adapté pour un individu souffrant d'un désordre de la peau, notamment de l'épiderme, selon la présente invention, caractérisé en ce qu'à l'étape a), le fragment de peau utilisé est un fragment issu d'une collection ou de culture de peau. d) the selection of said compound if the modifications obtained are those desired for the treatment. In a sixth aspect, the present invention relates to a method for determining the therapeutic treatment adapted for an individual suffering from a disorder of the skin, in particular of the epidermis, according to the present invention, characterized in that at the step a), the skin fragment used is a fragment from a collection or skin culture.
Sous un septième aspect, la présente invention a pour objet l'utilisation d'un modèle selon la présente invention, pour déterminer pour un produit les effets secondaires sur l'épiderme, tels que par exemple : In a seventh aspect, the present invention relates to the use of a model according to the present invention, for determining for a product the side effects on the epidermis, such as for example:
- sa toxicité ; - its toxicity;
- son adsorption ou absorption ; - its adsorption or absorption;
- sa distribution, son métabolisme, son excrétion ; - its distribution, its metabolism, its excretion;
- son pouvoir irritant ou corrosif ; - its irritating or corrosive power;
- son pouvoir sensibilisant. - its sensitizing power.
Un tel modèle pourra être en effet selon la présente invention être utilisé ou mis en œuvre dans tout procédé ou test in vitro ou ex vivo nécessitant des expérimentations animales ou humaines, comme par exemple l'étude de libération ou de pénétration de principes actifs, et/ou de leur biodisponibilité cutanée, l'étude de leur efficacité, ou encore de leur tolérance, de leur compatibilité, ces principes actifs étant à visée cosmétiques, dermatologiques et/pharmaceutiques. Such a model may indeed be used according to the present invention to be used or implemented in any method or test in vitro or ex vivo requiring animal or human experiments, such as, for example, the release or penetration study of active principles, and or of their cutaneous bioavailability, the study of their effectiveness, or even of their tolerance, of their compatibility, these active principles being for cosmetic, dermatological and pharmaceutical purposes.
Selon un mode de réalisation préféré, ledit produit est un produit cosmétique, dermatologique ou pharmaceutique. According to a preferred embodiment, said product is a cosmetic, dermatological or pharmaceutical product.
L'utilisation comme modèle concernera également sa mise en œuvre pour l'étude de toute pathologie se traduisant par des anomalies de la peau, notamment de l'épiderme mais également pour toutes les cellules situées sous l'épiderme pour lesquelles l'épiderme constitue une barrière naturelle et pour lesquelles une thérapie par voie topique est envisageable. The use as a model will also concern its implementation for the study of any pathology resulting in abnormalities of the skin, in particular of the epidermis, but also for all the cells located under the epidermis for which the epidermis constitutes a natural barrier and for which topical therapy is feasible.
D'autres caractéristiques et avantages de l'invention apparaissent dans la suite de la description avec l'exemple et la figure. La figure dont les légendes sont ci-après, ainsi que l'exemple qui suit sont destinés à illustrer l'invention sans pour autant en limiter la portée. LEGENDES DE LA FIGURE Other features and advantages of the invention appear in the following description with the example and the figure. The figure whose captions are below, as well as the following example are intended to illustrate the invention without limiting its scope. LEGENDS OF THE FIGURE
Figure : Schéma décrivant les différentes étapes du procédé Figure: Diagram describing the different steps of the process
- Etape 1 : Une biopsie cylindrique de peau est réalisée ; Step 1: A cylindrical skin biopsy is performed;
- Etape 2 : La biopsie est placée dans un container contenant une solution enzymatique dégradant la membrane basale ; - Step 2: The biopsy is placed in a container containing an enzymatic solution degrading the basement membrane;
- Etape 3 : La biopsie est incubée à 4 °C jusqu'à ce que le derme se détache de l'épiderme ; Step 3: The biopsy is incubated at 4 ° C until the dermis detaches from the epidermis;
- Etape 4 : L'épiderme est disposé sur une solution de plasma sanguin ; Step 4: The epidermis is placed on a solution of blood plasma;
- Etape 5 : La solution de plasma sanguin est solidifiée par coagulation; et Step 5: The blood plasma solution is solidified by coagulation; and
- Etape 6 : L'épiderme disposé sur le plasma coagulé est maintenu en culture. Step 6: The epidermis disposed on the coagulated plasma is maintained in culture.
EXEMPLE : Différentes étapes du procédé (voir figure) 1. Une biopsie cylindrique est tout d'abord réalisée sur un fragment de peau fraîchement prélevé pour permettre sa survie ex vivo. EXAMPLE: Different steps of the process (see figure) 1. A cylindrical biopsy is first performed on a fragment of skin freshly removed to allow its survival ex vivo.
2. La biopsie cutanée est délicatement déposée dans une solution enzymatique de dispase, de trypsine ou de thermo lysine, à environlO mg/ml de solution qui dégrade spécifiquement les protéines de la membrane basale, de façon à ce que la partie superficielle (l'épiderme) reste émergée alors que le derme sous- jacent est immergé. 2. The skin biopsy is gently deposited in an enzyme solution of dispase, trypsin or thermolysin, at about 10 mg / ml of solution which specifically degrades the basement membrane proteins, so that the superficial part (the epidermis) remains emerging while the underlying dermis is submerged.
3. L'ensemble est laissé à incuber (généralement au moins 10 heures à 4°C) jusqu'à ce que le derme s'enfonce progressivement dans la solution après s'être détaché de l'épiderme resté , quand à lui, étalé, flottant à la surface de la solution, et toujours associées à ses annexes. La séparation entre l'épiderme et le derme peut être achevée à l'aide de pinces en veillant à préserver l'intégrité des tissus. 3. The whole is allowed to incubate (usually at least 10 hours at 4 ° C) until the dermis gradually sinks into the solution after being detached from the epidermis remained, when to him, spread , floating on the surface of the solution, and always associated with its annexes. The separation between the epidermis and the dermis can be completed using forceps while ensuring the integrity of the tissues.
4. La solution enzymatique est ensuite remplacée par une solution dérivée de plasma sanguin traité avec un agent anti-coagulant aux propriétés réversibles en présence d'ions calcium (citrate de sodium). Cette solution contient 42 % de plasma sanguin, 50 % d'une solution de NaCl à 0,9 %, 8 % d'une solution saline de CaC12 à 1% et un agent anti-fibrinolytique (acide tranaxémique ou aprotinine). 5. En coagulant, le plasma fait office de support dermique sur lequel adhère l'expiant d'épiderme. La coagulation consiste essentiellement en la transformation, en présence d'ions calcium et de thrombine, du fïbrinogène présent dans le plasma en un échafaudage de molécules de fibrine reliées entre elles par des liaisons covalentes. L'agent anti-fibrinolytique a pour fonction d'inhiber les enzymes susceptibles de dégrader la matrice de plasma sécrétées par l'expiant d'épiderme, et ainsi de maintenir l'intégrité du support dermique. De la même façon la solution de plasma peut être substituée par une solution de fïbrinogène ou de collagène. Pour cette dernière, l'incubation à 37°C seule permet la solidification. 4. The enzymatic solution is then replaced by a solution derived from blood plasma treated with an anti-coagulant agent with reversible properties in the presence of calcium ions (sodium citrate). This solution contains 42% blood plasma, 50% 0.9% NaCl solution, 8% 1% CaCl 2 saline and an anti-fibrinolytic agent (tranaxemic acid or aprotinin). 5. By coagulating, the plasma acts as a dermal support on which adheres the epidermis explant. Coagulation consists essentially of the transformation, in the presence of calcium ions and thrombin, of the fibrinogen present in the plasma into a scaffold of fibrin molecules linked together by covalent bonds. The anti-fibrinolytic agent functions to inhibit the enzymes capable of degrading the plasma matrix secreted by the epidermis explant, and thus to maintain the integrity of the dermal support. In the same way, the plasma solution may be substituted with a solution of fibrinogen or collagen. For the latter, incubation at 37 ° C alone allows solidification.
6. L'ensemble constitué par l'expiant d'épiderme est maintenu en culture avec l'épidémie au contact de l'air. 6. The complex consisting of the explant of epidermis is kept in culture with the epidemic in contact with the air.
Claims
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR1059160A FR2967163A1 (en) | 2010-11-05 | 2010-11-05 | IN VITRO OR EX VIVO METHOD FOR SURVIVAL MAINTENANCE AND GENERATION OR RECONSTRUCTION OF AN EPIDERM |
| FR1059160 | 2010-11-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2012059703A1 true WO2012059703A1 (en) | 2012-05-10 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/FR2011/052587 WO2012059703A1 (en) | 2010-11-05 | 2011-11-07 | In vitro or ex vivo method for storing and/or keeping an epidermis alive |
Country Status (2)
| Country | Link |
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| FR (1) | FR2967163A1 (en) |
| WO (1) | WO2012059703A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013164436A1 (en) | 2012-05-03 | 2013-11-07 | Genoskin | System for keeping alive and transporting skin biopsies and applications of said system |
| FR3078541A1 (en) * | 2018-03-05 | 2019-09-06 | Genoskin | EX VIVO SUB-CUTANEOUS INJECTION MODEL |
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| EP0285474A1 (en) | 1987-03-26 | 1988-10-05 | Centre International De Recherches Dermatologiques Galderma - Cird Galderma | Skin equivalent |
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2010
- 2010-11-05 FR FR1059160A patent/FR2967163A1/en not_active Withdrawn
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2011
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| WO2013164436A1 (en) | 2012-05-03 | 2013-11-07 | Genoskin | System for keeping alive and transporting skin biopsies and applications of said system |
| FR2990106A1 (en) * | 2012-05-03 | 2013-11-08 | Genoskin | SYSTEM FOR SURVIVAL MAINTENANCE AND TRANSPORT OF SKIN BIOSPSIES AND ITS APPLICATIONS |
| US9585381B2 (en) | 2012-05-03 | 2017-03-07 | Genoskin | System for keeping alive and transporting skin biopsies and applications of said system |
| FR3078541A1 (en) * | 2018-03-05 | 2019-09-06 | Genoskin | EX VIVO SUB-CUTANEOUS INJECTION MODEL |
| WO2019170281A1 (en) * | 2018-03-05 | 2019-09-12 | Genoskin | Ex vivo subcutaneous injection model |
| KR20200128124A (en) * | 2018-03-05 | 2020-11-11 | 제노스킨 | Ex vivo subcutaneous injection model |
| JP2021516542A (en) * | 2018-03-05 | 2021-07-08 | ゲノスキン | Exobibo subcutaneous injection model |
| JP7361039B2 (en) | 2018-03-05 | 2023-10-13 | ゲノスキン | Ex vivo subcutaneous injection model |
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