Classifications

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  • 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
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  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/38—Vitamins
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/90—Polysaccharides
  • C12N2501/905—Hyaluronic acid
• • C—CHEMISTRY; METALLURGY
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  • 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
  • C12N2502/091—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells melanocytes
• • 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
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  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/04—Screening or testing on artificial tissues
  • C12N2503/06—Screening or testing on artificial skin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/54—Collagen; Gelatin

Definitions

• the present invention relates to an in vitro skin equivalent, in particular animal skin, preferably mammalian and / or human, and to its use.
• the present invention finds application particularly in the pharmacological, cosmetological and dermatological fields.
• references in brackets ([]) refer to the list of references at the end of the text.
• the skin is a very complex organ comprising a very particular stratified structure. It consists of three main parts:
• epidermis a superficial part, the thinnest, called epidermis
• hypodermis a deeper layer, the hypodermis.
• the skin naturally ensures the protection of the body while ensuring communication between it and the external environment.
• the skin is the first defense organ vis-à-vis any aggression.
• the aggressions suffered by the skin are multiple, it may be for example UV-related aggressions likely to cause inflammatory reactions / cellular alterations causing cancer, physical attacks such as burns, scarifications, for example due to blunt objects, chemical aggression, for example related to chemicals, for example detergents. These different aggressions can induce in particular skin disorders likely to change the structure of the skin, to alter its color, to cause the appearance of skin wounds and / or cause "aging" skin.
• Other elements may be at the origin of an alteration of the skin, for example genetic parameters and / or related to endocrine disorders and / or autoimmune systemic. This may include diseases causing a pigment disorder such as vitiligo, hypermelanosis, hypomelanosis or a nevus.
• systems / processes for preparing known skin substitutes use, in particular, media comprising compounds which are incompatible with clinical use, for example pituitary extract of bovine.
• the equivalent substitutes obtained can not, in particular, be used clinically or as a skin model.
• step b seeding a matrix comprising collagen with fibroblasts from step a;
• fibroblasts seeded in the matrix comprising collagen in a fibroblast culture medium M2 comprising ascorbic acid or an ascorbate or a derivative thereof, the matrix and cultured fibroblasts forming a dermal substitute;
• step g culturing the dermal substitute seeded in step g in a M5 skin culture medium thus forming the skin substitute.
• the term “include” may include “include”, “contain” or “encompass” on the one hand, or “constitute” or “on the other”.
• the dermal substitute obtained according to the process of the invention is a complete tissue reproducing the characteristics of an in vivo dermis, namely comprising macromolecules of the protein type, in particular collagen fibers, glycosaminoglycans, proteins and fibroblasts. functional.
• the dermis equivalent is also referred to as a dermis substitute.
• the skin equivalent obtained according to the process is advantageously a complete tissue reproducing the characteristics of an in vivo skin, namely comprising a keratinized pluristratified epithelium comprising keratinocytes reproducing a stratum basai, a stratum spinosum, a stratum granulosum and a histologically normal stratum corneum, basal melanocytes in contact with a dermal substitute containing functional fibroblasts via a functional basal lamina.
• the skin equivalent comprises a basal lamina consisting in particular of a protein mixture secreted by the cells of the substitute thus forming a dermal-epidermal junction reproducing the characteristics of a skin in vivo.
• the fibroblasts, melanocytes, keratinocytes that may be used in the process for obtaining the skin equivalent can all be fibroblasts, melanocytes, keratinocytes known to those skilled in the art. It may be, for example, fibroblasts, melanocytes and / or keratinocytes obtained from cell banks, for example from the National Collection of
• CNCM Microorganism Culture
• Type Culture Collection (ATCC), the company LGC Standards Sarl It can also be fibroblasts, melanocytes and / or keratinocytes isolated from a biological sample of an animal, preferably a mammal and / or a human being, previously isolated.
• the fibroblasts, melanocytes and / or keratinocytes may be fibroblasts, melanocytes, keratinocytes isolated independently of a biopsy or several biopsies.
• the fibroblasts, melanocytes and / or keratinocytes can be isolated independently of a biopsy or several biopsies of an individual, preferably a mammal and / or a human being, for transplanting said skin substitute onto said patient. It can be independently of fibroblasts, melanocytes and / or autologous or heterologous keratinocytes of an individual.
• the fibroblasts, melanocytes and / or keratinocytes can be isolated independently of a biopsy or several biopsies from, for example, Caucasian, Asian or African skin, different anatomical sites, for example back, face, breast, back of the hands , palms of a human being.
• fibroblasts, melanocytes and / or keratinocytes isolated independently of skin biopsies, preferably from a human being. It may be, for example, fibroblasts, melanocytes and / or keratinocytes independently isolated from healthy skin or having at least one cutaneous pathology. It may be, for example, fibroblasts, melanocytes and / or keratinocytes independently isolated from skin having, for example, age spots (actinic lentigo), melasma, vitiligo, nevus, melanoma, xeroderma pigmentosum.
• fibroblasts, melanocytes and / or keratinocytes isolated independently from mammalian skin biopsies exhibiting genetic pathologies, for example from human skin biopsy with progeria, restrictive dermopathy, epidermolysis bullosa, of ichthyosis. It may also be fibroblasts, melanocytes and / or keratinocytes independently isolated from mammalian skin biopsies under treatment pharmacological, for example it may be fibroblasts, melanocytes and / or keratinocytes isolated from a biopsy of a human under treatment against leukemia, a human under dermatological treatment, for example for treatment acne, juvenile acne.
• fibroblasts melanocytes and / or keratinocytes
• retroviruses lentiviruses
• adenoviruses adeno associated viruses
• AAV adeno associated viruses
• fibroblasts, melanocytes and / or keratinocytes independently overexpressing at least one protein, for example a protein chosen from collagen VII, keratins 5, 14, catalase, SIRT6 and / or undergoing at least one expression. protein, for example via the technique of small hairpin RNA (shRNA) or small interfering RNA (siRNA), for example collagen VII, HIF1, CCN3.
• shRNA small hairpin RNA
• siRNA small interfering RNA
• fibroblasts may be for example independently genetically modified fibroblasts, melanocytes and / or keratinocytes as described in Pendaries V et al, JID 2012 [1]; Petek LM et al Mol ther 2010 [2]. It may be for example fibroblasts, melanocytes and / or keratinocytes that are independently genetically modified or not, for example the Ker-CT cell identified under the reference ATCC CRL-4048, the TelCOFS02MA cell identified under the reference ATCC CRL-4005.
• fibroblasts may also be fibroblasts, melanocytes and / or keratinocytes derived from cell line, for example the HaCaT keratinocyte line, the fibroblast line WS1.
• fibroblasts melanocytes and / or keratinocytes independently obtained from adult stem cells, induced pluripotent stem cells, for example by maintaining said adult stem cells and / or induced pluripotent stem cells via, for example, introduction of genes Oct3 / 4, Sox 2, KLF4, c-Myc then differentiated by means of factor cocktails, for example retinoic acid and / or BMP-4, into a cell line.
• factor cocktails for example retinoic acid and / or BMP-4
• fibroblasts melanocytes and / or keratinocytes obtained by the method described in Kogut et al. Mol Biol Methods 2014 [3], in Ohta et al, Mol Biol Methods, 2013 [4], and / or in Revilla et al., Tissue Eng Regen Med Med, 2015 [5].
• M1 fibroblast culture medium means any medium known to those skilled in the art suitable for culturing fibroblasts. It may be, for example, a commercially available medium, for example a Dulbecco modified medium minimalized medium (DMEM) medium marketed by the company Gibco comprising in particular a mixture of amino acids, vitamins, inorganic salts of sugars, for example glucose, or a Fibrolife medium marketed by the company Cell Systems
• DMEM Dulbecco modified medium minimalized medium
• Gibco comprising in particular a mixture of amino acids, vitamins, inorganic salts of sugars, for example glucose, or a Fibrolife medium marketed by the company Cell Systems
• DMEM Dulbecco Medium Modified Eagle Medium Minimal Medium
• the M1 medium may further comprise supplements, including fetal calf serum (FCS).
• supplements including fetal calf serum (FCS).
• the M1 medium may comprise, for example, from 5 to 15% by weight, from 7.5 to 12.5% by weight, and 10% by weight of fetal calf serum (FCS) relative to the total weight of the medium. .
• FCS fetal calf serum
• the M1 medium may comprise at least one anti-fungal and / or antibiotic compound. It may be for example any anti-fungal and / or antibiotic compound known to those skilled in the art and / or commercially available. It may be for example at least one antifungal compound selected from the group comprising Amphotericin B, ketoconazole and a mixture thereof. It may be for example at least one antibiotic compound selected from the group consisting of penicillin, streptomycin, ciprofloxacin and a mixture thereof.
• the M1 medium may comprise from 0.1 to 10% by weight, from 0.5 to 5% by weight, 1% by weight of antifungal relative to the total weight of the medium.
• the M1 medium may comprise from 0.1 to 10% by weight, from 0.5 to 5% by weight, 1% by weight of antibiotics relative to the total weight of the medium.
• M1 and / or all of its constituents may be of clinical grade.
• clinical grade is meant herein that the component or medium has been recognized by the competent authority as being suitable for clinical use in a given territory.
• the medium is of clinical grade it does not include pituitary extract of cattle.
• step a. fibroblast culture can be carried out at a temperature of 30 to 40 ° C, 35 to 39 ° C, equal to 37 ° C.
• the fibroblast culture time of step a. can be 5 to 21 days, 5 to 15 days, 8 to 15 days.
• the fibroblast culture time of step a. can be carried out under a controlled atmosphere comprising 5 to 10% CO2, for example in an atmosphere comprising at least 5% CO 2 .
• step a. fibroblast culture can be carried out in an oven at a temperature of 30 to 40 ° C, from 32 to 40 ° C, equal to 37 ° C and under a controlled atmosphere containing at least 5% CO 2
• Fibroblast culture can be carried out in any suitable culture container known to those skilled in the art. It can be a petri dish, a culture bottle with a capacity of 25, 75 or 175 cm 2 .
• fibroblasts obtained by culture according to step a. may form a confluent cell mat in the culture container.
• the fibroblasts can be 70 to 100% confluent, preferably 100% confluent.
• the method may furthermore comprise:
• the step of the elimination of the culture medium can be carried out by any method known to those skilled in the art. It may be for example a suction of the medium, a flipping of the container to eliminate the culture medium.
• step a 'rinsing the cells can be carried out by any method known to those skilled in the art, for example, by soaking, spraying, incubating the cells in a rinsing solution.
• rinsing solution any cell rinsing solution known to those skilled in the art. It may be, for example, a Hank's Balanced Sait Solution (HBSS) buffer solution at a pH of between 7.2 and 7.4.
• HBSS Hank's Balanced Sait Solution
• PBS phosphate buffered saline
• Hank's a balanced solution of Hank's marketed respectively by the company Gibco, Sigma Aldrich, Lonza.
• the step of removing the rinsing solution can be carried out by any method known to those skilled in the art. It may be for example an aspiration of the rinsing solution, a flipping of the container to remove the rinsing solution.
• the trypsinization step can be performed by immersing the cells in a buffer solution (ST) comprising trypsin, followed by the addition of Fetal Calf Serum (FBS) in order to stop the enzymatic reaction.
• ST buffer solution
• FBS Fetal Calf Serum
• the buffer solution (ST) can be any buffer solution known to those skilled in the art that can be used in a trypsinization process. It may be for example a buffer salt phosphate (PBS), a balanced solution from Hank's marketed respectively by the company Gibco, Sigma Aldrich, Lonza.
• PBS buffer salt phosphate
• the amount of trypsin added in the buffer solution (ST) can be between 0.01 and 0.05% by weight relative to the total weight.
• the incubation time in the buffer solution comprising trypsin before addition of the SVF in the buffer solution (ST) can be between 2 and 10 min.
• the amount of SVF added in the buffer solution (ST) can be from 5 to 20% by volume relative to the total volume.
• the etching step can be carried out by any method known to those skilled in the art, such as sedimentation or centrifugation at a speed of 800 to 1400 rpm. for example equal to 1200 revolutions per minute.
• the centrifugation step may be carried out for a period of 4 to 10 minutes, for example 5 minutes.
• the centrifugation step can be carried out by any device known to those skilled in the art, for example a rotary centrifuge marketed by the companies Eppendorf or Jouan.
• matrix comprising collagen any matrix comprising collagen known to those skilled in the art that can be seeded by cells. It may be for example a collagen matrix corresponding to an unstretched type I collagen gel, preferably not imposing preferential fibroblast organization as described in Bell et al, 1979 [6]. It may be for example a matrix with a density / concentration of collagen, preferably type I collagen, with a surface area of 25 to 500 cm 2. It may be, for example a matrix comprising collagen available commercially, for example it may be a matrix comprising collagen marketed by Integra.
• the matrix comprising collagen may be a dermal regeneration matrix.
• the dermal regeneration matrix may be chosen in particular from the matrices sold under the names Integra (registered trademark) and Matriderm (registered trademark) by Integrara Life Science Corporation and MedSkin Solutions Dr. Suwelack AG respectively.
• the dermal regeneration matrices such as those mentioned above are already modeled, which promotes the reconstruction of the skin equivalent.
• the matrix comprising collagen may be a matrix comprising crosslinked collagen and at least one glycosaminoglycan, for example chondroitin 6-sulfate. It may be, for example, the Integra Matrix (trademark) marketed by Integralife Sciences and / or the matrix obtained according to the process described in document Boyce ST et al. 1988 [7].
• the matrix comprising collagen may be a matrix comprising collagen fibers of native structure and elastin.
• collagen fibers of native structure is meant especially non-chemically crosslinked fibers.
• This may be, for example, the Matriderm matrix marketed by MedSkin Solutions Dr. Suwelack AG and / or the matrix obtained according to the method described in document Hafemann et al, Burns 1999 [8].
• the thickness of the matrix comprising collagen may be from 1.0 to 3.0 mm (inclusive) before seeding by the fibroblasts. In a particular embodiment, the thickness of the matrix comprising collagen may be strictly greater than 1.0 mm before seeding by the fibroblasts.
• the seeding of step b can be carried out by any method known to those skilled in the art. It can be for example a application, for example by spraying a culture medium comprising the fibroblasts on the matrix, by deposition by transplanting the cells onto the matrix, by pouring a culture medium comprising the cells in suspension, for example by 3D printing such that described in Wonhye Lee et al. "Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform manufacturing.” Biomaterials, 2009, March; 30 (8): 1587-95 [7].
• step a comprises step a "
• the method of the invention may comprise, prior to step b of seeding, a step b1 of resuspending the cells centrifuged in the medium M1.
• the inoculation of step b of a collagen matrix can be carried out at a density of 20,000 to 50,000 fibroblasts / cm 2 , preferably 30,000 fibroblasts / cm 2 of surface area of the matrix comprising collagen.
• the density of fibroblasts may be strictly less than 50,000 fibroblasts / cm 2 of matrix comprising collagen.
• the fibroblast culture medium M2 may be any medium known to those skilled in the art suitable for culturing fibroblasts. This may be, for example, a commercially available medium, for example a Dulbecco Modified Eagle's Minimal Essential Medium medium (DMEM) comprising in particular a mixture of amino acids, vitamins, inorganic salts of sugars, for example glucose.
• DMEM Dulbecco Modified Eagle's Minimal Essential Medium medium
• the M1 medium may further comprise supplements, including fetal calf serum (FCS).
• supplements including fetal calf serum (FCS).
• the M2 medium may comprise from 5 to 15% by weight, from 7.5 to 12.5% by weight, 10% by weight of fetal calf serum (FCS) relative to the total weight of the medium.
• FCS fetal calf serum
• the medium M2 may comprise at least one antifungal and / or antibiotic compound. It may be for example any anti-fungal and / or antibiotic compound known to those skilled in the art and / or commercially available. For example, it may be at least an antifungal compound selected from the group consisting of Amphotericin B, ketoconazole and a mixture thereof. It may be for example at least one antibiotic compound selected from the group consisting of penicillin, streptomycin, ciprofloxacin and a mixture thereof.
• the M2 medium may comprise from 0.1 to 10% by weight, from 0.5 to 5% by weight 1% by weight of antifungal relative to the total weight of the medium.
• the medium M2 may comprise from 0.1 to 10% by weight, from 0.5 to 5% by weight, equal to 1% by weight of antibiotics relative to the total weight of the medium.
• the medium M2 may further comprise ascorbic acid or ascorbate.
• the M2 medium may comprise ascorbic acid or ascorbate at a concentration of 20 to 60 mg.mL -1 , for example 30 to 55 mg.mL -1 , equal to 50 mg.mL -1 .
• ascorbic acid makes it possible in particular to promote the remodeling of the matrix comprising collagen by stimulating the synthesis of collagen by fibroblasts.
• M2 medium and / or all of its constituents may be of clinical grade.
• step c. fibroblast culture can be carried out at a temperature of 30 to 40 ° C, 35 to 39 ° C, equal to 37 ° C.
• the culture time of step c. fibroblasts can be 5 to 12 days, 7 to 10 days.
• the culture of step c. fibroblasts can be carried out under a controlled atmosphere comprising at least 5% CO2.
• step c. culturing fibroblasts seeded in the matrix comprising collagen may comprise: a first step c 'of culture of 18 to 28 hours in the presence of a medium M2 1 of fibroblast culture comprising neither ascorbic acid nor ascorbate, and
• a second culturing step of at least 2 days in the presence of a fibroblast culture medium M2 2 comprising ascorbic acid or an ascorbate.
• the fibroblast culture medium M2 1 corresponds to the medium M2 as defined above comprising neither ascorbic acid nor ascorbate.
• the present medium M2 1 and / or all of its constituents may be of clinical grade.
• the fibroblast culture medium M2 2 corresponds to the medium M2 as defined above comprising ascorbic acid or an ascorbate.
• the medium M2 2 and / or all of its constituents may be of clinical grade.
• step c '. Culture can be carried out at a temperature of 30 to 40 ° C, 35 to 39 ° C, equal to 37 ° C.
• the culture time of step c '. can be from 19 to 27 hours, for example 24 hours
• the cultivation step c can be carried out at a temperature of 30 to 40 ° C, 35 to 39 ° C, equal to 37 ° C.
• the cultivation time of step c1 may be from 5 to 12 days, equal to 7 days.
• the inventors have also advantageously demonstrated that the cultured matrix and fibroblasts obtained in step c. form a structure corresponding to a dermis substitute.
• step c 'of culture corresponds to a step of adhesion and colonization of the matrix by the fibroblasts and step c "advantageously allowing a remodeling of the matrix comprising the fibroblasts to form a dermal substitute.
• M2 1 and M2 2 will advantageously form a dermis substitute wherein the fibroblasts do not proliferate but colonize the matrix comprising collagen while advantageously allowing the production of collagen by the fibroblasts themselves, thus allowing a remodeling of the dermis.
• the product obtained at the end of step c. can be advantageously used as a substitute dermis.
• this product includes all the physicochemical characteristics of the dermis from which the fibroblasts can be derived.
• step d Melanocyte culture can be carried out in any suitable culture container known to those skilled in the art. It can be a petri dish, a culture bottle with a capacity of 25 to 75 cm 2 , 25, 75 of 125 cm 2 .
• the melanocyte culture medium M3 can be any medium known to those skilled in the art suitable for melanocyte culture. It may be for example a commercially available medium, for example a medium commercially available under the reference "Melanocyte Medium M2", "MBM” marketed by the company Promocell, in a MCDB medium marketed by the company. Sigma-Aldrich company including a mixture of amino acids, vitamins, inorganic salts of sugars, for example glucose, as shown in Table 3 below:
• modified medium for example the MCDB153 medium further comprising complementary amino acids, for example tyrosine, methionine or a mixture thereof, additional inorganic salts for example sodium bicarbonate (NaHCOs).
• complementary amino acids for example tyrosine, methionine or a mixture thereof
• additional inorganic salts for example sodium bicarbonate (NaHCOs).
• the M3 medium may also comprise at least one complement chosen from bovine pituitary extract (BPE), insulin, penicillin streptomycin (PS), hydrocorticosone, horse serum, and calf serum. , basic fibroblast growth factor (bFGF), granulocyte macrophage stimulating factor (GM-CSF), SCF or any mixture thereof.
• BPE bovine pituitary extract
• PS penicillin streptomycin
• hydrocorticosone horse serum
• calf serum calf serum
• bFGF basic fibroblast growth factor
• GM-CSF granulocyte macrophage stimulating factor
• SCF any mixture thereof.
• the M3 medium may comprise from 0.1 to 10%, by weight, of 0.5 to 5% by weight, 1% by weight of penicillin streptomycin (PS) based on the total weight of the medium.
• PS penicillin streptomycin
• the medium M3 can comprise a concentration of 1.25 to 1.60 ⁇ , 1.40 to 1.55 ⁇ , 1.45 ⁇ of hydrocortisone.
• the M3 medium may comprise a concentration of 100 to 160 Mg.mL -1 , of 1 to 150 Mg.mL -1 , equal to 140 pg.mL -1 bovine pituitary extract (BPE).
• BPE bovine pituitary extract
• the M3 medium may comprise a concentration of 15 to 25 gmL -1 , equal to 20 gmL -1 insulin.
• the M3 medium may comprise a concentration of 0.01 to 0.2 Mg.mL “1 , of 0.01 to 0.1 Mg.mL “ 1 , equal to 0.01 M9- " L " 1 from GM-CSF
• the M3 medium may comprise a concentration of 0.004 to 0.2 Mg.mL- 1 , of 0.01 to 0.15 Mg.mL- 1 , equal to 0.05 Mg.mL- 1 of SCF.
• the M3 medium may comprise a concentration of 0.1 to 10 ng.mL -1 , 0.5 to 5 ng.mL -1 , 0.8 to 2 ng.mL -1 , equal to 1 ng.mL "1 of bFGF.
• the medium M3 may comprise from 1 to 5% by weight, from 2 to 4% by weight, 3% by weight of horse or calf serum relative to the total weight of the medium.
• M3 and / or all of its constituents may be of clinical grade.
• Melanocyte culture can be carried out at room temperature, for example at a temperature of 30 to 40 ° C, for example equal to 37 ° C.
• the culture time of step d. can be 15 to 28 days.
• the culture of step d. melanocytes can be carried out under a controlled atmosphere comprising at least 5% CO 2 .
• the melanocytes obtained by culture according to step d. may form a confluent cell mat in the culture container.
• melanocytes can form a cell mat of 50 to 100% confluency.
• the method may furthermore comprise:
• the step of removing the culture medium can be carried out by any method known to those skilled in the art. It may be for example a suction of the medium, a flipping of the container to eliminate the culture medium.
• the step of rinsing the cells can be carried out by any method known to those skilled in the art, for example, by spraying, soaking the cells in a rinsing solution.
• melanocyte rinsing solution any melanocyte rinsing solution known to those skilled in the art. It may be for example an HBSS buffer solution, for example of the solution described in Table 2 above, of phosphate buffered saline (PBS) with a pH of between 7.2 and 7.4. It may also be a commercially available buffer solution, for example a phosphate buffered saline (PBS), a balanced solution of Hank marketed respectively by the company Gibco, Sigma Aldrich, Lonza.
• PBS phosphate buffered saline
• the step of removing the rinsing solution can be carried out by any method known to those skilled in the art. It may be for example an aspiration of the rinsing solution, a flipping of the container to remove the rinsing solution.
• the trypsinization step can be performed by immersing cells in a buffer solution (ST) comprising trypsin, followed by the addition of Fetal Calf Serum (FBS) in order to stop the reaction. enzyme.
• ST buffer solution
• FBS Fetal Calf Serum
• the buffer solution (ST) may be a buffer solution as defined above.
• the amount of trypsin added in the buffer solution (ST) may be 0.01 to 0.05% by weight relative to the total weight.
• SVF in the buffer solution can be 2 to 5 min.
• the amount of FBS added to the solution (ST) can be from 5 to 20% by volume based on the total volume.
• the "pelletizing" step can be carried out by sedimentation, by centrifugation by any method known to the man of the job. It can be for example a centrifugation at a speed of 800 to 1200 revolutions per minute.
• the centrifugation step can be carried out for a period of 5 to 10 minutes.
• the centrifugation step may be carried out by any device known to those skilled in the art, for example a rotary centrifuge marketed by the companies Eppendorf or Jouan.
• the centrifugation step makes it possible to sediment the cells in order to separate them from the medium.
• the person skilled in the art by his general knowledge, will be able to adapt / modify the centrifugation step by any means. known technique for sedimenting cells in a medium.
• Keratinocyte culture can be carried out in any suitable culture container known to those skilled in the art. It can be a petri dish, a culture bottle with a capacity of 25 to 75 cm 2 , 25, 75 or 125 cm 2 .
• the M4 keratinocyte culture medium may be any medium known to those skilled in the art suitable for culturing keratinocytes. It may be, for example, a commercially available medium, for example a KSFM medium marketed by the life-technology company, KGM sold by the company lonza, provitro in a MCDB 153 medium marketed by Sigma-Aldrich comprising in particular a mixture of amino acids, vitamins, inorganic salts of sugars, for example glucose.
• KSFM medium marketed by the life-technology company
• KGM sold by the company lonza
• provitro in a MCDB 153 medium marketed by Sigma-Aldrich comprising in particular a mixture of amino acids, vitamins, inorganic salts of sugars, for example glucose.
• MCDB153 medium comprising a concentration of sodium chloride of 0.100 to 0.1 10 M / L, for example 0.104 M / L, a concentration of Hepes 2 to 3x10 "2 M / L, for example 2.29 x 10" 2 M / L, a concentration of sodium bicarbonate 1, 10 x 10 "2 M / L to 1 x10 25" 2 M / L, for example
• the M4 medium may further comprise supplements selected from growth factors, for example epithelial growth factor (EGF), bovine pituitary extract (BPE), insulin, penicillin-streptomycin (PS ), hydrocortisone or any mixture thereof.
• the M4 medium may comprise clinical grade supplements. These may be, for example, supplements chosen from growth factors, for example epithelial growth factor (EGF), insulin, penicillin-streptomycin (PS), hydrocorticosone or any mixture thereof. .
• the M4 medium may comprise for example 0.5 to 5% by weight, 0.75 to 3% by weight, 1% by weight penicillin-streptomycin (PS) based on the total weight of the medium.
• PS penicillin-streptomycin
• the M4 medium may comprise a concentration of 1.25 to 1.60 .mu., from 1.40 to 1.55 .mu.l, of 1.45 .mu.l of hydrocortisone.
• the M4 medium may comprise a concentration of 50 to 90 gmL -1 , 60 to 80 gmL -1 , 70 gmL -1 bovine pituitary extract.
• the M4 medium may comprise a concentration of 3 to 8 g.mL -1 , for example equal to 5 gmL -1 insulin.
• the M4 medium may comprise a concentration of 5 to 15 ng.mL -1 , 6.5 to 13 ng.mL -1 , equal to 10 ng.mL -1 of epithelial growth factor (EGF).
• EGF epithelial growth factor
• M4 medium and / or all of its constituents may be of clinical grade.
• step e. The keratinocyte culture can be carried out at a temperature of 25 to 39 ° C, for example equal to 37 ° C.
• the culture time of step e. can be from 15 to 28 days,
• the culture of step e. keratinocytes can be carried out under a controlled atmosphere comprising at least 5% CO 2 .
• the cultured keratinocytes according to step e. can form a monolayer of cells in the culture container. It may be for example a monolayer of cells close to confluence, for example from 50 to 80% confluence in the culture container.
• the method may furthermore comprise:
• a step e 'of removing the culture medium, rinsing the cells with a solution, removing the rinsing solution,
• step e of detachment of the cells by trypsinization, and a step e '" of centrifugation.
• step e the elimination of the culture medium can be carried out by any method known to those skilled in the art. It may be for example an aspiration of the medium, a reversal of the container in order to eliminate the culture medium.
• step e 'rinsing cells can be carried out by any method known to those skilled in the art, for example, by soaking, spraying, incubation of cells in a keratinocyte rinsing solution.
• keratinocyte rinsing solution any keratinocyte rinsing solution known to those skilled in the art. It may be for example a PBS, HBSS buffer solution, for example as described in Table 2 above, at a pH of between 7.2 and 7.4. It can also be a commercially available buffer solution, for example a phosphate buffered saline (PBS), a balanced solution of Hank's marketed respectively by the company Gibco, Sigma Aldrich, Lonza.
• PBS phosphate buffered saline
• step e the removal of the keratinocyte rinsing solution can be carried out by any method known to those skilled in the art. It may be for example an aspiration of the keratinocyte rinsing solution, a reversal of the container in order to eliminate the keratinocyte rinsing solution.
• the trypsinization step e can be performed by immersing the cells in a solution (S) comprising trypsin, followed by the addition of Fetal Calf Serum (FBS) in order to stop the enzymatic reaction.
• S a solution
• FBS Fetal Calf Serum
• the amount of trypsin added in the solution (S) can be from 0.01 to 0.05% relative to the total weight of the solution.
• the incubation time of the trypsin before addition of the SVF in the medium can be from 5 to 10 min.
• the amount of FBS added in the solution (S) can be from 5 to 20% by weight relative to the total weight of the solution.
• the centrifugation step may be carried out by any method known to those skilled in the art, such as centrifugation at a rate of 800 to 1200 rpm.
• the centrifugation step can be carried out for a period of 5 to 10 minutes.
• the centrifugation step may be carried out by any device known to those skilled in the art, for example a rotary centrifuge marketed by the companies Eppendorf or Jouan.
• step f. mixture of melanocytes obtained in step d. with keratinocytes obtained in step e. can be achieved by any suitable process means known to those skilled in the art. It may be for example a mixture of cells with stirring in a culture medium.
• the mixture of melanocytes and keratinocytes of step f can be carried out with a ratio of melanocytes / keratinocytes in number of 1/20 to 11 ⁇ 5, equal to 1/19.
• the inventors have surprisingly demonstrated that when the mixture of melanocytes and keratinocytes is carried out with a melanocyte / keratinocyte ratio of 1/20 to 1/15, preferably equal to 1/19, the skin substitute or equivalent of obtained skin has structural / biological characteristics identical to those of an in-vivo skin.
• the mixture of melanocytes and keratinocytes of step f is carried out with a ratio melanocytes / keratinocytes in number of 1/20 to 1/15, equal to 1/19, and the matrix comprising collagen. is a dermal regeneration matrix as defined above.
• the seeding of the dermis substitute of step g can be carried out by any method known to those skilled in the art. This may be, for example, an application, for example by spraying the culture medium comprising a mixture of melanocytes and keratinocytes obtained in step f., By depositing by transplanting the cells onto the dermis substitute, by pouring drip of the culture medium comprising a mixture of melanocytes and keratinocytes obtained in step f, for example by 3D printing as described in Wonhye Lee et al. "Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform manufacture.” Biomaterials, 2009, Mar; 30 (8): 1587-95 [9].
• the seeding of the dermis substitute of step g can be advantageously carried out with a ratio (keratinocytes + melanocytes) / fibroblasts of 9 to 19.
• the inventors have in fact surprisingly demonstrated that when the seeding of step g is made with a ratio (keratinocytes + melanocytes) / fibroblasts from 9 to 19, the skin substitute or skin equivalent obtained has structural / biological characteristics identical to those of a normal skin.
• the seeding of the dermis substitute of step g is carried out with a ratio (keratinocytes + melanocytes) / fibroblasts of 9 to 19 and the matrix comprising collagen is a dermal regeneration matrix as defined. above.
• M5 skin culture medium means any medium known to those skilled in the art suitable for skin culture.
• This may be, for example, a commercially available medium, for example a modified green medium, namely comprising 2/3 medium "DulbeccoA / ogt modified Eagle's minimal essential medium” (DMEM); 1/3 of "Ham's F12” medium and comprising 10% of Fetal Calf Serum (FCS) commercially mixed mixture by Gibco including a mixture of amino acids, vitamins, inorganic salts, sugars, for example glucose.
• DMEM DulbeccoA / ogt modified Eagle's minimal essential medium
• FCS Fetal Calf Serum
• It may also be a modified green medium, that is to say a green medium free of cholera toxin, triodothyronine, or a mixture of the medium “Iscove's Modified Dulbecco's Medium” (IMDM) and the medium MCDB153 comprising 10% FCS; or an IMDM / "dermalife keratinocyte” mixture comprising 10% FCS marketed respectively by the company Gibco, Lifescience, Promocell and Sigma Aldrich.
• IMDM Iscove's Modified Dulbecco's Medium
• the M5 medium may also further comprise supplements selected from hyaluronic acid or hyaluronate or a derivative thereof, ascorbic acid or ascorbate or a derivative thereof, or a mixture of these
• the M5 medium may comprise, for example, from 40 to 60 mg.L -1 , from 45 to 55 mg.L -1 , 50 mg.L -1 of hyaluronate or hyaluronic acid.
• the M5 medium comprises hyaluronic acid or a hyaluronate and / or a derivative thereof it does not include pituitary extract of bovine.
• the M5 medium may comprise, for example, from 40 to 60 mg.L -1 , from 45 to 55 mg.L -1 , 50 mg.L -1 of ascorbic acid or ascorbate.
• step h. skin culture can be carried out at a temperature of 25 to 40 ° C, for example equal to 37 ° C.
• the skin culture time of step h. can be between 6 and 21 days, for example 8 to 15 days.
• the skin culture of step h. skin can be carried out under a controlled atmosphere comprising at least 5% CO 2 .
• the skin culture of step h. skin can be carried out at a temperature of 25 to 40 ° C, for example equal to 37 ° C and in a controlled atmosphere comprising at least 5% of CO2 .
• M5 medium and / or each of its components may be of clinical grade.
• step h. can understand:
• a first step h. at least 6 hours, preferably 6 to 24 hours, in the presence of a culture medium M5 1 comprising neither hyaluronic acid, hyaluronate, ascorbic acid nor ascorbate,
• a second step of 0-7 day culture preferably at least 2 days, in the presence of a culture medium M5 2 comprising hyaluronic acid or a hyaluronate or a derivative thereof, and a third step h.
• a culture medium M5 2 comprising hyaluronic acid or a hyaluronate or a derivative thereof
• a third step h "culture of at least 2 days in 3 M5 medium comprising hyaluronic acid or hyaluronate or a derivative thereof, and ascorbic acid or ascorbate or a derivative of these.
• the M5 1 medium of the skin substitute culture corresponds to the M5 medium as defined above comprising neither ascorbic acid nor ascorbate.
• M5 medium and / or each of its components may be of clinical grade.
• the M5 2 medium for a skin substitute culture or skin equivalent corresponds to the M5 medium as defined above comprising hyaluronic acid or a hyaluronate or a derivative thereof while being free of ascorbic acid or ascorbate or a derivative thereof.
• M5 2 may be a clinical grade medium.
• the M5 3 culture medium of skin substitute or skin equivalent corresponds to the M5 medium as defined above comprising hyaluronic acid or a hyaluronate or a derivative thereof and ascorbic acid or ascorbate or a derivative thereof.
• M5 3 may be a clinical grade medium.
• the duration of step h ', of culture can be from 6 to 24 hours, for example from 8 to 24 hours, from 12 to 18 hours.
• step h 'of skin culture can be carried out under a controlled atmosphere comprising at least 5% CO 2 .
• step h makes it possible to promote the adhesion of melanocytes and keratinocytes to the dermis substitute.
• the culture step h " can be carried out by depositing in a M5 2 culture medium the seeded dermis substitute obtained in step h 'or immersion or submersion of the seeded dermis substitute obtained in step h 'in an M5 culture medium 2 .
• the cultivation step h " can be carried out at a temperature of 25 to 40 ° C, for example equal to 37 ° C.
• the duration of the culture step may be from 0 to 7 days, preferably from 2 to 7 days.
• step h "of skin culture can be carried out under a controlled atmosphere comprising at least 5% CO 2 .
• the cultivation step h '" can be carried out by depositing in a M5 2 culture medium the seeded dermal substitute obtained in step h". Immersion of the seeded dermal substitute obtained in step h ", in a culture medium M5 2 , or immersion seeded dermis substitute obtained in step h"., said substitute being immersed in said medium to the air-liquid interface, or said medium being immersed in the middle flower.
• middle flower immersion of the substitute in the medium so as to cover the substitute over its entire height without immersion of its upper part.
• the cultivation step h '" can be carried out at a temperature of from 25 to 40 ° C, for example equal to 37 ° C.
• the duration of the culture step may be from 2 to 7 days, preferably 7 days.
• step h '"of skin culture can be carried out under a controlled atmosphere comprising at least 5% CO 2.
• the inventors have demonstrated that immersion of the seeded dermal substitute obtained in step h ", in an M5 3 culture medium comprising hyaluronic acid or a hyaluronate or a derivative thereof and the Ascorbic acid or an ascorbate or a derivative thereof promotes the formation of the dermal-epidermal junction and thus ensures a better polarization of the cells of the seeded dermal substitute.
• the inventors have also demonstrated that immersion up to the air-liquid or medium-flower interface of the substitute of seeded dermis obtained in step h ", in an M5 3 culture medium comprising hyaluronic acid or a hyaluronate or a derivative thereof and ascorbic acid or an ascorbate or a derivative thereof. a differentiation of the epidermis thus promoting the formation of a stratum corneum on the substitute or equivalent of skin.
• the inventors have also demonstrated that the immersion up to the air-liquid or medium-density interface of the seeded dermis substitute obtained in step h "in an M5 3 culture medium comprising hyaluronic acid. or a hyaluronate or a derivative thereof and ascorbic acid or an ascorbate or a derivative thereof allows maintenance of the high proliferative power of the basal layer cells and a decreasing gradient of proliferative power concomitant with the increased differentiation of the epidermis similar to the gradients observed in the skin in vivo.
• the inventors have also demonstrated that when the M5 medium comprises hyaluronic acid or a hyaluronate or a derivative thereof, this makes it possible, surprisingly, to improve the quality of the substitute or equivalent of skin comprising the set of constituent layers thereof with a structure identical to that of the skin in-vivo.
• the inventors have demonstrated that the method advantageously makes it possible to obtain a skin substitute or skin equivalent comprising all of the constituent layers thereof.
• the skin substitute obtained according to the process of the invention has characteristics similar to that of the native skin, in contrast to the skin substitutes known in the state of the art.
• the inventors have also demonstrated that the method makes it possible to obtain a dermal substitute and / or a substitute or equivalent of skin of a size much greater than those known in the state of the art.
• the method advantageously makes it possible to obtain a dermis substitute and / or a substitute or equivalent of skin with a surface area of 1 to 25 cm 2 , for example from 5 to 25 cm 2 .
• the inventors have also advantageously demonstrated that the method makes it possible to obtain a substitute or equivalent of dermis and / or an equivalent or substitute of skin with a minimum amplification capacity of 6.
• the inventors have also advantageously demonstrated that the method makes it possible to obtain an equivalent of dermis and / or skin that can be handled with viscoelastic properties advantageously making it possible to avoid any physical tearing / alteration of said equivalent during its handling.
• the skin equivalent comprises melanocytes at the level of the basal layer forming an epidermal unit of melanization.
• the skin equivalent obtainable by the aforementioned method, by the presence of melanocytes advantageously has a constitutive pigmentation.
• the present invention also relates to the use of a skin equivalent according to the invention as a laboratory tool.
• laboratory tool is meant the use, for example of the skin equivalent in in-vitro tests. It may be, for example, tests for studying the physiology, structure, evaluation of an agent, for example a pigmenting agent, photoprotective, antioxidant, on the skin equivalent. .
• the keratinocytes, melanocytes and fibroblasts included in the equivalent may be independently derived from different tissues / biopsies / cell cultures, thus the skin equivalent may advantageously be representative of a pathology and / or allow the study of the influence of a group of cells, pathological or not, on the other groups of cells present in the equivalent.
• the skin equivalent obtained may be representative and / or be a model of a cutaneous pathology, for example juvenile acne, vitiligo, melasma, senile lentigo, scleroderma.
• the skin equivalent obtained may represent or be a model of pathological skin, for example a carcinoma-type skin model of melanoma.
• the present invention also relates to the use of a skin equivalent in a method of evaluating at least one candidate agent.
• the candidate agent may be a candidate compound and / or an external agent.
• the skin substitute can be used in any method of evaluating candidate compounds known to those skilled in the art and suitable for the evaluation of compounds vis-à-vis the skin.
• the skin equivalent can be used in the process described in US 2004/0015149, in US 5,882,248, in US 2008/0097607, in EP 2 781 191.
• skin equivalent means an equivalent and / or skin substitute as mentioned above.
• It can be any chemical and / or biological compound known to those skilled in the art. It may be for example a compound known to those skilled in the art and / or commercially available and / or a compound obtained by chemical synthesis, by extraction from plant material, bacterial and / or yeast. It may be for example a compound or cosmetic active, for example a compound or cosmetic active mentioned in the international dictionary of cosmetic ingredients ("International Cosmetic Ingredient Dictionary and Handbook"). It may also be a pharmaceutical and / or dermatological compound, for example any compound mentioned in the Merck Index and / or in the "Orange Book".
• external agent is understood to mean, for example, ultraviolet (UV) radiation, infrared (IR) radiation, hypoxia, hyperoxia, stale air, for example having a high concentration of carbon dioxide. and / or nitrogen monoxide, and / or polluted air, for example city.
• UV ultraviolet
• IR infrared
• hypoxia hypoxia
• hyperoxia hyperoxia
• stale air for example having a high concentration of carbon dioxide. and / or nitrogen monoxide, and / or polluted air, for example city.
• the evaluation method can also be a method of screening candidate compounds.
• the evaluation method may include the steps of:
• the step of contacting can be carried out by any method known to those skilled in the art. This may be for example a direct application on a part and / or the entire surface of the skin equivalent, by soaking the skin equivalent in a solution comprising said agent, by any means known to the person skilled in the art.
• the person skilled in the art by his general knowledge will adapt according to the candidate compound the contacting step with the equivalent of skin.
• the effect determining step can be carried out by any method and / or means known to those skilled in the art.
• This may be for example a comparison of the skin equivalent with an equivalent of control skin that has not been brought into contact with the agent.
• This may be, for example, a visual observation of the macroscopic appearance of the skin equivalent, observation under optical microscope of the skin equivalent possibly previously subjected to staining, immunohistochemistry, for the determination of melanin, DNA damage, epidermal differentiation. It may also be a proteomic or transcriptomic analysis of equivalents extracts treated or not treated with agents.
• the skin substitute has a size much greater than those known in the state of the art allowing in particular the concomitant evaluation of agents on the same equivalent.
• the dermis equivalent comprises at least one type I collagen matrix in which the fibroblasts are distributed. It may also contain other constituents of the extracellular matrix, for example molecules such as collagens, in particular collagen IV, laminins and glycosaminoglycans.
• Figure 1 shows a diagram of the steps for obtaining a substitute / equivalent of skin.
• FIG. 2 represents optical microscopy photographs of skin (FIG. 2A), skin substitutes / equivalents obtained according to the method with seeded ratio ratios of cells (FIGS. 2B and 2C).
• FIG. 3A is a photograph of a dermis substitute obtained in step c by optical microscopy after staining of the fibroblasts.
• Figure 3B is a photograph of a skin substitute obtained in small size ie 0.5cm 2
• Figure 3C is a photograph of a skin substitute obtained of average size ie 25 cm 2 .
• FIG. 4 represents optical microscopy photographs of skin equivalent obtained according to the method with a ratio of seeded cells (keratinocytes + melanocytes) / fibroblasts of 13.3 (FIGS. 4A and B); immunohistochemical staining of the basal melanocytes (FIG. 4C, light areas) and the production of the basal lamina, collagen IV staining (FIG. 4D (2)) and the proliferation marker p63 (FIG. 4D (1)).
• Example 1 Example of Manufacturing a Skin Substitute
• the cells used came from a skin biopsy performed at the level of mammoplasties previously carried out on a patient.
• the biopsy sample was taken by a plastic surgeon and the biopsy deposited in sterile tube containing saline.
• modified MCDB153 medium comprising the compounds listed in Table 4 below further comprising 5.88 g sodium bicarbonate / 5L, 0.272 g of tyrosine / 5L and 0.157 g of L Methionine / 5L, the pH of the medium being adjusted to 7.4
• differential trypsinization trypsinization with 0.025% trypsin and 0.01 M EDTA (1-2 minutes to detach the melanocytes, 10 minutes to detach the keratinocytes). The melanocytes take off first, which makes it possible to purify the cultures.
• fibroblasts Trypsinization of fibroblasts with 0.025% trypsin and 0.01 M EDTA for 10 minutes and then neutralization with irradiated FBS, centrifugation at 1200 rpm with a GR 2022 centrifuge for 5 minutes, and seeding in DMEM comprising 10% FBS on a dermal matrix of sterile collagenic origin, namely an Integra matrix (registered trademark) previously rinsed with a balanced saline solution of Hank (“Hank's Balanced Sait Solution" (HBSS) 3 times with 30,000 fibroblasts per cm 2 in a chamber of stainless steel incubation made to measure.
• HBSS Hank
• the seeded matrix was incubated at 37 ° C., 5% CO 2 in DMEM comprising 10% irradiated FBS and penicillin and 1% streptomycin and 50 mg / ml ascorbic acid for 1 week with change of medium. every 3 days.
• d Trypsinization of the keratinocytes and melanocytes with 0.025% trypsin and 0.01 M EDTA for 1 to 2 minutes to take off the melanocytes from the melanocyte culture dishes, then for 10 minutes to detach the keratinocytes from the keratinocyte culture dishes. The melanocytes took off first, which makes it possible to purify the cultures. Neutralization with irradiated SVF and centrifugation and seeding at 400,000 cells per cm 2 in an incubation chamber of a mixture containing 1 melanocyte per 19 keratinocytes.
• Figure 1 shows a diagram of the steps for obtaining a skin substitute.
• two skin equivalents obtained had ratios (keratinocytes + melanocytes) / fibroblasts respectively of 13.3.
• the quantities of cells seeded were respectively 15,000 for the fibroblasts, 10,000 for the melanocytes and 190,000 for the keratinocytes.
• FIG. 2B shows a photograph in optical microscopy of a skin substitute obtained according to the method in which the ratio (keratinocytes + melanocytes) / fibroblasts was 13.3.
• FIG. 2C represents an optical microscopy photograph of a skin substitute obtained according to the method in which the ratio (keratinocytes + melanocytes) / fibroblasts was 6.7 and FIG. 2 has an optical microscopy photograph of a biopsy of normal skin.
• FIGS. 4A and 4B also represent optical microscopy photographs of skin equivalent obtained according to the method in which the ratio (keratinocytes + melanocytes) / fibroblasts was 13.3.
• FIGS. 4E and 4F represent photographs in optical microscopy of skin in vivo after the immunohistochemical staining of the melanocytes in the basal position (FIG. 6E, light areas), the labeling of collagen IV (FIG. 4F (2)) and of the p63 marker 4F (1)) .It appears clearly in FIGS. 4C and 4D that the skin equivalent according to FIG. the invention comprises melanocytes, a basal lamina as demonstrated by the presence of collagen IV at which the cells present are highly proliferative as for the skin in vivo (FIGS. 4E and 4F).
• the substitute / skin equivalent obtained by the process has a structure identical to that of the skin in vivo.

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