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Definitions

• the present invention relates to a process for the in vitro preparation of a dermal papilla equivalent from fibroblasts derived from the dermal papilla and / or the conjunctive sheath.
• the present invention also relates to a process for the in vitro preparation of an equivalent of hair follicle by culturing proliferative epithelial cells on said dermal papillae thus obtained.
• the anagen phase is the active phase of the hair, the one during which it lives and grows regularly. It lasts from 4 to 7 years.
• the germ cells that surround the papilla of the bulb of the hair root continually make the material that allows the hair to live and grow. Then, the multiplication of the germinal cells stops at the bottom of the follicle.
• the active phase of the hair is then completed; another begins, which is much shorter.
• the catagen phase In just 15 days, the bulb of the hair disappears (because it is no longer fed because of the interruption of the germinal cells) and transforms at an accelerated speed.
• the papilla disappears, that is to say that the hollow bulb becomes full; it keratinizes, hardens and becomes horny. The hair is then dead; the follicle tightens to expel the dead hair.
• the telogen phase is the phase that lasts about three months and during which the dead hair is waiting to fall. In order to fall, the hair must be pushed out by the new hair which in turn grows in the same follicle and will expel the old one.
• Regeneration of the hair follicle is then made from stem cells, called germ cells located in the "bulge".
• the "bulge” is formed by a cell subpopulation of the outer epithelial sheath, called germinal cells, located in the middle portion of the hair follicle, and more exactly at the level of the insertion zone of the muscle arrector of the hair. These cells represent the lowest part of the permanent portion of the follicle.
• keratinocytes are relatively undifferentiated, biochemically and ultrastructurally.
• This "bulge” is in a strategic position to interact during the late telogen phase, with the ascending dermal papilla and initiate a new follicle in anagen.
• the germ cells are therefore essential cells for the renewal of the hair.
• the germinal cells (germinal cells or pluripotent cells or stem cells) of the telogen hair are involved in the exit of the dormant phase of the hair follicle and thus the regrowth of the hair.
• the hair bulb is pear-shaped and consists of:
• a papilla which is a budding of dermal origin, located at the base of the follicle. It is a highly vascularized site that participates in the nutrition and regulation of hair growth by its reserve of growth factors and extracellular matrix proteins. This information will be transmitted to the matrix cells, elaborated in the matrix, to allow their differentiation (Rees JL Genetics of hair and skin color).
• the matrix which is a zone capping the dermal papilla, consisting of a cluster of little differentiated matrix cells. It is the seat of intense mitotic activity.
• the cells of the matrix which are localized in the hair bulb and which form a small cell cluster around the dermal papilla, consist mainly of precursors of keratinocytes which constitute a germinative base and which proliferate rapidly to differentiate by forming the hair shaft. , thus playing a vital role in the hair cycle. From the beginning of the anagen phase to the end of it, these matrix cells will proliferate to the catagen phase and disappear into the telogen phase.
• the matrix also includes follicular melanocytes that are responsible for the pigmentation of the hair. Proliferation and differentiation of these matrix cells are controlled by the dermal papilla (Botchkarev VA, Ishimoto J. Molecular control of epithelial-mesenchymal interactions during hair follicle cycling.) J Invest Dermatol Symp Proc 2003 Jun; 8 (1): 46-55, Review).
• the outer epithelial sheath constitutes the outer envelope of the follicle: it is an invagination of the epidemic. This one houses especially stem cells from which the hair follicle will be cyclically regenerated.
• the inner epithelial sheath separates the outer epithelial sheath from the hair shaft. This sheath consists of three cell types organized concentrically keratinized layers that accompany the growth of the hair. We distinguish the Henle layer, the Huxley layer and the cuticle which is formed of flattened cells directed towards the hair matrix.
• the hair shaft which is partly visible, is the hair.
• the structure of the hair shaft consists of three distinct layers from the outside to the inside.
• the cuticle, the cortex and the medulla are all composed of keratinized cells.
• Alopecia is conditioned by various factors: genetic, hormonal and environmental, diet and physical activity. Hair has an essential aesthetic and identity role.
• human hair follicles are obtained by culturing different types of cells present in the hair bulb.
• the skilled person has cultured in vitro 2D or 3D hair follicle cells from different compartments that make up the bulb.
• Higgins has shown that in 2D in vitro culture the cells of the dermal papilla lose their induction capacity as soon as they are first cultured and amplified.
• the spheroids obtained in 2D and 3D culture described by Higgins do not exhibit the functional characteristics of a dermal papilla in vivo, in particular these spheroids have a low activity with alkaline phosphatase, Versican, and SFRP2 (secreted frizzled related protein 2) as shown in Example 6 below.
• none of the aforementioned models contain proliferative epithelial cells such as matrix cells or germ cells, the presence and amount of which are essential for the formation of a follicular structure.
• WO2017 / 055358 discloses the ability of the cells of the matrix to regenerate a micro hair follicle in the presence of ROCK inhibitor. Nevertheless, these cells of the matrix are seeded on fibroblasts having lost their capacity to proliferate (3T3 fibroblasts, human fibroblasts irradiated or treated with mitomycin), and not on dermal papillae. This implies functional and morphological differences as shown in Figure 10 below.
• the Applicant has demonstrated, on the one hand, that the culture of fibroblasts resulting from the dermal papilla and / or the conjunctive sheath in a serum-free medium on specific 2D or 3D culture media of the microplate type round, these supports do not allow the adhesion of the cells, allows to obtain an in vitro equivalent of dermal papilla having most of the functional and morphological characteristics of a dermal papilla vivo.
• the seeding of proliferative epithelial cells such as the cells of the matrix and / or the germinal cells on said in vitro equivalents of dermal papillae obtained makes it possible to obtain an in vitro equivalent of hair follicle having most of the characteristics functional and morphological of a human vivo follicle, and in particular likely to regenerate.
• the present invention relates to a process for the in vitro preparation of a dermal papilla equivalent comprising at least one step of culturing fibroblasts originating from the dermal papilla and / or the conjunctive sheath on a carrier comprising a serum-free nutrient culture medium B for a period of time sufficient to allow said fibroblasts to separate from said support and cluster to form at least one spheroid; the surface of said support used does not allow the adhesion of the cells; said culture medium is selected from 2D or 3D microplate round bottom culture media.
• a second subject of the present invention relates to an in vitro equivalent of a dermal papilla that can be obtained according to the aforementioned method.
• a third subject of the present invention relates to the use of a dermal papilla equivalent according to the invention and of proliferative epithelial cells such as the cells of the matrix and / or the germinal cells for the preparation of an in vitro equivalent. vitro hair follicle.
• the invention relates to a process for the in vitro preparation of a hair follicle equivalent comprising at least one step of culturing proliferative epithelial cells in the presence of at least one dermal papilla equivalent according to the invention during a period of time sufficient to allow differentiation of said proliferative epithelial cells into keratinocytes positive for K85 and K35 markers.
• the present invention also relates to an in vitro equivalent of hair follicle obtainable by the above method.
• the present invention also relates to the therapeutic uses of the hair follicle equivalent according to the invention in the prophylactic or therapeutic treatment of a state of reduced hair growth, and the treatment of alopecia.
• the invention relates to the use of the hair follicle equivalent according to the invention for the in vitro test of effects of active agents on hair properties, and in particular for the identification of compounds. modulator of the growth of hair and / or hair.
• the present invention also relates to a method for screening compounds modulating hair growth and / or hair using the equivalent of hair follicle according to the invention.
• fibroblasts derived from the dermal papilla fibroblasts taken from a microdissection of an anagen hair follicle at the budding of dermal origin located at the base of the hair follicle .
• fibroblasts resulting from the conjunctive sheath means fibroblasts taken from a microdissection of a hair follicle in the anagen phase at the base of the hair follicle under the dermal papilla.
• the fibroblasts originating from the dermal papilla or the conjunctive sheath are preferably taken from operating waste or biopsies, for example facelift waste or scalp removal because a simple pulling of the hair does not allow these cells to be recovered.
• the fibroblasts used in the context of the present invention are not fibroblasts whose proliferation has been previously stopped, preferably by having previously irradiated (for example, with gamma rays) or previously treated with mitomycin.
• the fibroblasts according to the invention are not 3T3 fibroblasts. Proliferative epithelial cells
• proliferative epithelial cells means epithelial cells present in the hair follicle and capable of giving rise to all the cell types present in a hair.
• the proliferative epithelial cells are preferably selected from matrix cells and / or germ cells.
• cells of the matrix refers to the cells located in the hair bulb and which form a small cell cluster around the dermal papilla (Ebling FJ, The biology of hair.Dermatol Clin, 1987 Jul; 5 (3): 467 81. Review, Saitoh M, Uzuka M, Sakamoto M. Human Hair Cycle, J Invest Dermatol, 1970 Jan, 54 (l): 65-81). These cells can be sampled, amplified and stored in tissue banks for later use.
• these cells may be removed by the following method: hair follicles are placed in a petri dish containing a minimum culture medium supplemented with 2% antibiotic and non-essential amino acids.
• the bulbar region is cut at the top of the dermal papilla and the epithelium of the bulb is separated from the dermal papilla and the conjunctive sheath.
• germinal cells means the stem cells present at the level of the "bulge”.
• germinal cells are removed in the telogen phase.
• telogen is understood to mean the follicle which contained the hair in the telogen phase.
• the germ cells can be removed by the following method: hair follicles in the telogen phase are placed in a petri dish containing a minimum culture medium supplemented with 2% antibiotic and non-essential amino acids.
• the germ cell region is extracted from the connective sheath and then placed in a Petri dish containing a feeder layer of 3T3i fibroblasts. This microdissection technique thus makes it possible to preserve the quantity and the integrity of the cells, because they are not separated from each other.
• the present invention relates to a process for the in vitro preparation of a dermal papilla equivalent comprising at least one step of culturing fibroblasts originating from the dermal papilla and / or the conjunctive sheath. on a support comprising a serum-free nutrient culture medium B for a period of time sufficient to allow said fibroblasts to separate from said support and cluster to form at least one spheroid; the surface of said support used does not allow the adhesion of the cells; said culture medium is selected from 2D or 3D microplate round bottom culture media.
• spheroid means a 3D microtissu in the form of a sphere in which the cells will be organized and synthesize the extracellular matrix.
• the term "serum-free" means a culture medium comprising less than 5% by volume of serum, less than 4% by volume, less than 3% by volume, less than 2% by volume. less than 1% by volume, preferably 0% by volume of serum relative to the total volume of the culture medium.
• “Serum” is understood to mean the serum proteins contained in the serum, and in particular at least one serum protein chosen from albumins, globulins such as alpha-globulins, alpha 2-globulins, betaglobulins, gamma globulins, and their mixtures.
• albumins such as alpha-globulins, alpha 2-globulins, betaglobulins, gamma globulins, and their mixtures.
• clusters refers to a grouping of cells in 2D.
• aggregates or “early spheroid” means a cluster of 3D cells of irregular shape, unorganized and not yet synthesizing extracellular matrix.
• said nutrient culture medium B comprises from 500 to 1500 mg / L of amino acids, from 2 to 18 mg / L of vitamins, from 1500 to 4500 mg / L of glucose, from 8750 to 10000 mg / L of inorganic salts, from 2 to 20 ⁇ g / ml of insulin, from 2 to 60 ng / ml of hydrocortisone, and optionally from 50 to 200 ⁇ g / ml of antibiotics and / or antimycotics.
• said nutrient culture medium B comprises from 600 to 1250 mg / L of amino acids, from 5 to 15 mg / L of vitamins, from 1750 to 2250 mg / L of glucose, from 9000 to 9750 mg / L of salts.
• said nutrient culture medium B comprises from 700 to 1150 mg / L of amino acids, from 5 to 15 mg / L of vitamins, from 1750 to 2250 mg / L of glucose, from 9000 to 9750 mg / L of inorganic salts, from 5 to 15 ⁇ g / ml of insulin, from 5 to 50 ng / ml of hydrocortisone, and optionally from 100 to 180 ⁇ g / ml of antibiotics and / or antimycotic.
• the amino acids present in said medium B are preferably chosen from glycine, L-glutamine, L-alanine, L-arginine, L-asparagine-H20, L-aspartic acid, L-cysteine, L-cystine 2HC1, L- glutamic acid, L-histidine, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, disodium salt dihydrate of L -tyrosine, L-valine, and mixtures thereof.
• the vitamins present in said medium B are preferably chosen from ascorbic acid, biotin, choline chloride, D-calcium pantothenate, ergocalciferol, folic acid, menadione sodium bisulfate, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, retinyl acetate, vitamin B12, alpha tocopherol disodium phosphate, i-inositol, and mixtures thereof.
• the inorganic salts present in said medium B are preferably chosen from anhydrous calcium chloride (CaCl2), copper sulphate pentahydrate (CuSO4-5H2O), ferrous sulphate heptahydrate (FeSO4-7H2O), anhydrous magnesium sulphate (MgSO4), sulphate of manganese monohydrate (MnSO4-H20), potassium chloride (KO), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), anhydrous sodium dihydrogenphosphate (NaH2PO4), zinc sulfate heptahydrate (ZnSO4-7H2O), and their mixtures.
• CaCl2 calcium chloride
• CuSO4-5H2O copper sulphate pentahydrate
• FeSO4-7H2O ferrous sulphate heptahydrate
• MgSO4 anhydrous magnesium sulphate
• MnSO4-H20 sulphate of manganese monohydrate
• K potassium chloride
• antibiotics present in said medium B include penicillin, streptomycin, and mixtures thereof.
• antimycotics present in said medium B include amphotericin B.
• Said serum-free nutrient culture medium B preferably comprises less than 15 ⁇ g / ml of growth factors, preferably less than 12 ⁇ g / ml of growth factors.
• the growth factors present in said nutrient medium B are selected from insulin and / or hydrocortisone.
• said nutrient culture medium B comprises from 80% to 99% by volume of Williams E medium, from 250 to 350 mg / L of glutamine, from 2 to 20 ⁇ g / ml of insulin, from 2 to 60 ng / ml of hydrocortisone, and optionally from 50 to 200 ⁇ g / ml of antibiotics and / or antimycotic.
• said nutrient culture medium B comprises from 95% to 98% by volume of Williams E medium, from 280 to 320 mg / L of glutamine, from 5 to 15 ⁇ g / ml of insulin, from 5 to 50 ng / ml of hydrocortisone, and optionally from 100 to 180 ⁇ g / ml of antibiotics and / or antimycotic.
• the Williams E medium is in particular free of glutamine.
• the fibroblasts are preferably cultured for at least 3 days, more preferably between 4 and 21 days.
• the fibroblasts are preferably seeded at high density.
• a density of at least 3000 cells / cm 2 preferably at least 9000 cells / cm 2 , more preferably from minus 14000 cells / cm 2 , even more preferably in a density of between 14000 cells / cm 2 and 47600 cells / cm 2 .
• the fibroblasts are seeded in a density of between 23,500 cells / cm 2 and 24,500 cells / cm 2 , more preferably in a density of 23,800 cells / cm 2 .
• the support that does not allow the adhesion of cells, in particular fibroblasts, is chosen from 2D and 3D microplate round-bottom culture media.
• the 2D and 3D supports used in the context of the present invention are not coated with collagen.
• bacteriological petri dishes not treated for cell culture, in particular plastic, not allowing the adhesion of cells.
• the surface of said support is preferably neutral.
• neutral surface in the sense of the present invention, an uncharged surface.
• the surface of said support may be hydrophobic or hydrophilic, preferably hydrophobic.
• hydrophilic it is covered with a substance such that it prevents any cellular adhesion, in particular, such a substance may be chosen from hydrogels covalently bonded to the surface of said support.
• the surface of said support is neutral and hydrophobic.
• a bacteriological petri dish sold under the name Falcon® sold by the company Corning (reference: 351007) may be used.
• round-bottomed microplate 3D culture media a round-bottom 96-well microplate marketed under the name of costar® by Corning (reference: 7007) may be used.
• the 3D culture support is not a flat-bottomed microplate.
• said fibroblasts derived from the dermal papilla and / or the sheath are seeded on a 2D culture support that does not allow the adhesion of the cells to a density of at least 14000 cells / cm 2 , preferably in a density between 14000 cells / cm 2 and 47600 cells / cm 2 , more preferably between 23500 cells / cm 2 and 24500 cells / cm 2 .
• said fibroblasts derived from the dermal papilla and / or the sheath are seeded on a round-bottomed microplate 3D culture support that does not allow the cells to adhere in a density of at least 3000 cells.
• the process for the in vitro preparation of a dermal papilla equivalent according to the invention may furthermore comprise a preliminary step of amplifying said fibroblasts resulting from the dermal papilla and / or fibroblasts resulting from the conjunctive sheath in a nutrient culture medium A; said nutrient culture medium A comprising at least serum, in particular fetal calf serum.
• the culture support used for this amplification step is a support treated to allow adhesion of the cells.
• these supports are those conventionally used for cell culture and are therefore well known to those skilled in the art.
• said nutrient culture medium A used in said amplification step comprises from 500 to 2000 mg / L of amino acids, from 15 to 35 mg / L of vitamins, from 2500 to 4500 mg / L of glucose, from 7500 to 9500 mg / L of inorganic salts, 5% to 30% by volume of fetal calf serum, and possibly 50 to 200 ⁇ g / ml of antibiotics and / or antimycotics.
• said nutrient culture medium A used in said amplification step comprises 750 to 1800 mg / L of amino acids, 20 to 30 mg / L of vitamins, 3000 to 4000 mg / L of glucose, of 8000 at 9000 mg / L of inorganic salts, from 10% to 25% by volume of fetal calf serum, and optionally from 100 to 180 ⁇ g / ml of antibiotics and / or antimycotic agents.
• the amino acids present in said culture medium A used in said amplification step are preferably chosen from glycine, L-alanyl-glutamine, L-arginine hydrochloride, L-cystine 2HCl, L-histidine hydrochloride-H 2 O, L isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L- valine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and mixtures thereof.
• the vitamins present in said culture medium A used in said amplification step are preferably chosen from choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, i- inositol and their mixtures.
• the inorganic salts present in said culture medium A used in said amplification step are preferably chosen from calcium chloride (CaCl 2 ), magnesium sulphate (MgSO 4), iron nitrate (Fe (NO 3 ) 9H 2 0), potassium chloride (KCl), sodium bicarbonate (NaHCO 3 ), sodium chloride (NaCl), sodium dihydrogenphosphate (NaH 2 PO 4, 4H 2 O), and mixtures thereof.
• antibiotics present in said medium A include penicillin, streptomycin, and mixtures thereof.
• said nutrient culture medium A used in said amplification step comprises from 70% to 80% by weight. volume of DMEM Glutamax medium, from 5% to 25% of fetal calf serum, from 50 to 90 mg / L of nonessential amino acids, and possibly from 50 to 200 ⁇ g / ml of antibiotics and / or antimycotics . Even better, said nutrient culture medium A comprises 78% by volume of DMEM Glutamax medium, 20% of fetal calf serum, 60 to 80 mg / L of nonessential amino acids, and optionally from 100 to 180 ⁇ g / ml antibiotics and / or antimycotics.
• the nonessential amino acids present in said culture medium A used in said amplification step are selected from L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and their mixtures.
• the process for preparing an in vitro equivalent of the dermal papilla according to the invention further comprises, prior to the step of culturing said fibroblasts, the following preliminary steps: a, isolate a hair follicle in the anagen phase from a scalp sample; b recover the fibroblasts from the dermal papilla and / or the conjunctive sheath using a microdissection of the dermal papilla and / or the conjunctive sheath;
• FCS fetal calf serum
• the present invention also relates to an in vitro equivalent dermal papilla obtainable by the method according to the invention as described above.
• the dermal papilla equivalent according to the invention is characterized in that it exhibits a positive alkaline phosphatase activity, and optionally a positive expression of the proteins chosen from BMP2 (Bone morphogenetic protein 2) and SFRP2 (secreted frizzled related protein 2), CORIN, SOX2, VCAN (Versican) and / or PLC (Perlecan).
• the dermal papilla equivalent according to the invention is preferably characterized in that it consists of cells originating from a step of culturing fibroblasts originating from the dermal papilla and / or the conjunctive sheath on a support comprising a serum-free nutrient culture medium B for a period of time sufficient to allow said fibroblasts to separate from said support and cluster to form at least one spheroid; said support not allowing the adhesion of the cells; said culture medium is selected from 2D or 3D microplate round bottom culture media.
• the dermal papilla equivalent according to the invention is in particular a sphere of diameter ranging from ⁇ to 250 ⁇ .
• the dermal papilla equivalent according to the invention is a sphere of approximately 200 ⁇ m in diameter.
• the enzymatic activity of the alkaline phosphatase can for example be measured by the NBT / BCIP alkaline phosphatase kit marketed by the Roche Laboratory (ref: 11 681 451 001 at October 30, 2017) where the BCIP (5-Bromo-4- salt) chloro-3-indolyl phosphate toluidine), substrate of alkaline phosphatase, will first be dephosphorylated and then oxidized to give a blue colored product.
• the protein expression of the markers BMP2, SFRP2, CORIN, SOX2, VCAN and PLC can for example be measured using the immunofluorescence technique, a technique which is also well known to those skilled in the art. Process for the preparation of a hair follicle equivalent
• the present invention further relates to the use of an in vitro equivalent of dermal papilla according to the invention and proliferative epithelial cells for the preparation of an in vitro equivalent of hair follicle.
• the present invention also relates to a process for the in vitro preparation of a hair follicle equivalent comprising at least one step of culturing proliferative epithelial cells in the presence of at least one dermal papilla equivalent according to the invention for a sufficient period of time to allow differentiation of said proliferative epithelial cells into keratinocytes positive for K85 and K35 markers.
• the proliferative epithelial cells are preferably chosen from the cells of the matrix, the germinal cells as defined above, and their mixtures.
• the proliferative epithelial cells used in the context of the present invention are the cells of the matrix.
• the proliferative epithelial cells seeded on at least one dermal papilla equivalent according to the invention are cultured at 37 ° C. and 5% CO 2 in a defined medium, such as that described by Philpott (1993).
• the proliferative epithelial cells are cultured in the same nutrient culture medium as that used for the preparation of the dermal papilla equivalents according to the invention, in particular the serum-free nutrient culture medium B.
• said dermal papilla equivalent is obtained by the method of preparing dermal papilla equivalent as mentioned above in the "Process for the preparation of a dermal papilla equivalent".
• the step of culturing said proliferative epithelial cells in the presence of at least one dermal papilla equivalent according to the invention is preferably carried out on 2D or 3D supports as defined above, the surface of which does not allow cell adhesion.
• the proliferative epithelial cells are preferably seeded at high density.
• the proliferative epithelial cells are seeded in a density of at least 2000 cells / cm 2 , preferably at least 6000 cells / cm 2 , and even more preferably in a density of between 6000 and 10,000 cells / cm 2 .
• said proliferative epithelial cells are cultured for a period of time sufficient to allow the formation of at least one tubular structure, also called follicular organoid.
• tubular structure or “follicular organoid” means the budding observed after culturing at least one dermal papilla equivalent according to the invention, associated with proliferative epithelial cells chosen from the cells of the womb. and / or germ cells.
• Said proliferative epithelial cells are cultured in the presence of a dermal papilla equivalent for at least 3 days, preferably for at least 5 days, even more preferably between 5 and 20 days, preferentially between 5 and 15 days.
• the process for preparing the hair follicle equivalent according to the invention comprises a step of adding to the nutrient culture medium B growth factors selected from the WNT family proteins, in particular the WNT3A protein, the proteins of the BMP family, in particular the BMP2 and BMP4 proteins, and their mixtures. Adding said growth factors is achieved between 1 day and 5 th day after seeding the proliferating epithelial cells.
• B growth factors selected from the WNT family proteins, in particular the WNT3A protein, the proteins of the BMP family, in particular the BMP2 and BMP4 proteins, and their mixtures. Adding said growth factors is achieved between 1 day and 5 th day after seeding the proliferating epithelial cells.
• the present invention also relates to an in vitro equivalent of hair follicle obtainable by the aforementioned method.
• the in vitro equivalent of hair follicle is characterized in that it consists of a dermal papilla equivalent with positive alkaline phosphatase activity and keratinocytes positive for markers K85 and K35 and possibly Ki67.
• the alkaline phosphatase activity is measured according to the method mentioned in the paragraph "Method for preparing a dermal papilla equivalent".
• the markings of keratin specific to hair K85, K35 and protein Ki67 marker of cell proliferation are carried out according to the immunofluorescence method.
• the hair follicle equivalent has in particular a tubular structure with a diameter of between ⁇ and 250 ⁇ , and a length of at least 500 ⁇ , preferably of length ranging from 500 ⁇ to 2500 ⁇ , more preferably from 1500 ⁇ to 2500 ⁇ .
• the process for the in vitro preparation of a hair follicle equivalent according to the invention may comprise, prior to the step of culturing proliferative epithelial cells in the presence of at least one dermal papilla equivalent according to the invention, a preliminary step of amplification of proliferative epithelial cells in the presence of an effective amount of a ROCK inhibitor.
• the proliferative epithelial cells such as the cells of the matrix and the germinal cells are extracted by microdissection of the hair follicle and are amplified according to the technique of Rheinwald and Green (Cell, vol 6, 331-344, 1975) by culture on a feeder support consisting of fibroblasts in a suitable medium known to those skilled in the art, in the presence of growth factors, including amino acids, serum, cholera toxin, insulin, tri-iodothyronine and pH buffer solution.
• growth factors including amino acids, serum, cholera toxin, insulin, tri-iodothyronine and pH buffer solution.
• such a culture medium may in particular contain at least one mitogenic growth factor for keratinocytes (for example epidermal growth factor (EGF) and / or keratinocyte growth factor (KGF), in particular KGF), insulin , hydrocortisone and optionally an antibiotic (eg, gentamycin, amphotericin B) to which a Rock inhibitor has been added.
• the ROCK inhibitors may be selected from: Y27632, Ripasudil, Fasudil, Thiazovivin and mixtures thereof.
• said medium may further comprise serum or a pituitary extract, for example of bovine origin, epinephrine, transferrin and / or nonessential amino acids.
• serum or a pituitary extract for example of bovine origin, epinephrine, transferrin and / or nonessential amino acids.
• the fibroblasts used for this culture will more preferably be 3T3 fibroblasts.
• 3T3 fibroblasts are well known to those skilled in the art. It is a fibroblast cell line known since 1962. "3T3" means "3-day transfer, inoculum 3xl0 5 cells".
• the culture of the proliferative epithelial cells is preferably a culture on fibroblasts (preferentially fibroblasts 3T3) whose proliferation has been previously stopped, preferably by having previously irradiated (for example, gamma rays) or previously treated with mitomycin. Mitomycin (in particular, mitomycin C) blocks the proliferation of these cells without preventing them from producing nutrients useful for the proliferation of keratinocytes.
• the effective amount of the ROCK inhibitor in particular Y27632, is between 1 and 100 ⁇ and preferably between 5 and 25 ⁇ and preferably ⁇ .
• the epithelial cells are cultured in the presence of the ROCK inhibitor, in particular Y27632, for at least 2 days and preferably for at least 3 days.
• the cells are cultured at a cell density of between 1000 and 4000 cells / cm 2 and preferably at a density of 3000 cells / cm 2 .
• the in vitro equivalents of dermal papilla and hair follicle according to the invention will therefore also find applications for the preparation of implants and / or skin substitutes to treat a skin disorder such as a burn, a defect in healing or canitie .
• a therapeutic effect is defined as a return to the normal state of hair, whether totally or partially.
• the prophylactic treatment is recommended if the subject has a prerequisite for hair loss, as a family predisposition.
• the conditions of a reduced amount of hair can be the result of alopecia, hereditary baldness, scarring, burns or accidental injury.
• the present invention also relates to a hair follicle equivalent according to the invention for the prophylactic or therapeutic treatment of a reduced hair condition.
• Another of its objects is a hair follicle equivalent according to the invention for the treatment of alopecia.
• the present invention also relates to a dermal papilla equivalent for the prophylactic or therapeutic treatment of a reduced hair condition or alopecia.
• the dermal papilla and hair follicle equivalents according to the invention make it possible, in particular, to carry out growth kinetics of the hairs or hair and therefore any study requiring many live and as complete hair as possible in an in vivo context such as the study of the cycle. hair and factors that can influence this cycle up to the study of assets promoting hair growth, assets to fight against hair loss or assets slowing growth of hair.
• the present invention further relates to the use of an in vitro equivalent of hair follicle according to the invention for the identification of compounds modulating the growth of hair and / or hair.
• the present invention also relates to a method for screening at least one hair and / or hair growth modulator compound comprising a step (a) of bringing said test compound into contact with an in vitro equivalent of a follicle. hair according to the invention then a step (b) of analyzing the effect of said compound on at least one parameter of the in vitro equivalent of hair follicle and a step (c) of selecting the compound modifying said parameter.
• the modulating compound to be tested is applied topically, for example, formulated in conventional topical formulations or introduced into the culture medium.
• Stage (b) may, in particular, be carried out by analyzing the expression, production and / or activity of markers related to the quality and / or homeostasis of the hair follicle as per for example epidermal and / or dermal markers, such as structural proteins.
• markers related to the quality and / or homeostasis of the hair follicle as per for example epidermal and / or dermal markers, such as structural proteins.
• structural proteins can be mentioned keratin hair.
• step (b) of the screening method the effect of the product on the growth of the hair shaft will be analyzed in step (b) of the screening method.
• the step (b) of analyzing the effect of the product will preferably be a comparison of at least one parameter measured on the equivalent of the hair follicle brought into contact with the test product to that measured on a control hair follicle equivalent grown under the same conditions but which did not receive the product to be tested.
• the step (c) of selecting the product modifying the parameter of the hair follicle equivalent will be based on a criterion determined in advance.
• the modification of this parameter may be a stimulation, a decrease or a total or partial inhibition of the expression, the production and / or the activity of said markers and / or the growth of the hair shaft.
• the criterion for selecting said product will be, for example, that this product has a stimulating or inhibiting effect on the parameter measured.
• the hair follicle equivalent according to the invention can also be used in automated methods for screening cosmetic, pharmaceutical or dermatological compounds to identify new active agents.
• the invention also relates to a method for screening at least one hair and / or hair growth modulator compound comprising a step (a) of bringing said test compound into contact with an in vitro equivalent.
• dermal papilla according to the invention then a step (b) of analyzing the effect of said compound on at least one parameter of the in vitro equivalent of dermal papilla and a step (c) of selecting the compound modifying said parameter.
• FIG. 1 Location of the cells of the matrix and fibroblasts used in the context of the present invention.
• Figure 2 Location of germ cells.
• Figure 3 Amplification of fibroblasts derived from dermal papilla in the nutrient culture medium A.
• Figure 4 Obtaining dermal papilla equivalent after culturing amplified fibroblasts in serum-free nutrient culture medium B.
• Figure 6 Formation of a tubular structure at the origin of the formation of an equivalent of hair follicle (T + 4 days from the seeding of the cells of the matrix on the dermal papillae equivalents).
• Figure 7 Growth of the equivalent of hair follicle (T + 10 days from the seeding of the cells of the matrix on the dermal papillae equivalents).
• Figure 8 Highly positive alkaline phosphatase activity of dermal papilla equivalent according to the invention.
• Figure 9 Hair follicle with positive labeling for markers K35, K85 and Ki67.
• Figure 10 Comparative WO2017 / 055358: T + 10 days from the seeding of the cells of the matrix.
• FIG. 11 Comparative Excludes Invention: Dermal papilla obtained according to the method described in Example 2 of WO 2009/118283 (magnification x10, J6)
• Figure 13 Comparative except invention: hair follicle cyst obtained according to the method described in Example 3 of WO 2009/118283 (J3).
• Figure 14 Comparative out-invention: culture of fibroblasts derived from dermal papilla on a 2D culture medium for cell culture (i.e. support for the adhesion of cells).
• FIG. 15 Dermal papilla equivalent according to the invention obtained according to Example 1 on 3D microplate round bottom culture medium (J2).
• Figure 16 Comparative out of invention: hair follicle obtained in 3D collagen gel culture.
• Example 1 Preparation of a dermal papilla equivalent according to the invention
• the nutrient culture medium A for amplification of fibroblasts has the following composition:
• Iron Nitrate Fe (N0 3) 3 "9H 2 O) 0.1
• Nutrient culture medium B for the preparation of the dermal papilla equivalents has the following composition:
• Disodium salt dihydrate of L-Tyrosine 50.65 Compounds Concentration (mg / L) l.-Valine 50.0
• Zinc Sulphate (ZnS04-7H20) 2.01.-4
• the dermal papilla located in the bulbar region of the follicle is located under the microscope. Using a scalpel and needles, the dermal papilla is microdissected and placed in a culture dish containing the nutrient culture medium A as described above. ( Figure 3). Culture-Preparation of the dermal papilla equivalent After amplification of the fibroblasts in monolayer culture, the fibroblasts are trypsinized and then deposited in an untreated petri dish for cell culture (Falcon® bacterial bacteriological box, Corning, ref: 351007) with a high density (for example 23800 cells per cm 2 ), in the serum-free nutrient culture medium B as described above.
• Fibroblasts migrate into the petri dish and cluster to form clusters, then cell aggregates, to finally detach from the support to form spheroids or dermal papillae equivalents after 5 days of culture.
• the dermal papillae obtained are spherical in shape of about 200 ⁇ m in diameter.
• Labeling of the alkaline phosphatase enzyme activity is carried out using the NBT / BCIP alkaline phosphatase kit (Roche ref: 11,681,451,001) where the BCIP (5-Bromo-4-chloro-3-indolyl salt) toluidine phosphate), substrate of alkaline phosphatase, will first be dephosphorylated and then oxidized to give a blue colored product.
• the dermal papillae obtained are also spherical in shape of about 200 .mu.m in diameter (see FIG. 15) and have a strongly positive alkaline phosphatase enzymatic activity.
• the dermal papillae according to the invention thus obtained have the morphological and functional characteristics of a dermal papilla in vivo, Example 2 - Preparation of a Hair Follicle Equivalent According to the Invention Experimental Protocol i. Microdissection of the cells of the matrix
• the hair follicles are extracted from a surgical scalp residue.
• the latter is first cut into portions of 5 mm 2 and then cut with a scalpel between the dermis and the hypodermis.
• the follicles are extracted using ophthalmic surgery forceps and are then cut just above the disc with a scalpel.
• the bulb is then recovered.
• the bulb has two compartments: the dermal compartment (dermal papilla and conjunctive sheath) and the cells of the matrix which form a cellular mass. ( Figure 1).
• the growing conditions have three main components:
• DMEM + 10% FCS + 7F medium (called G7F medium) has the following composition:
• FCS Fetal calf serum
• Epidermal growth factor 10 ng / ml
• T3 Triiodothyonine
• Adhesion Surface The cells of the matrix adhere and proliferate in the Green base medium in the presence of a feeder layer of 3T3 murine fibroblasts arrested in the cell cycle by mitomycin treatment.
• the cells are recovered at the confluent stage by enzymatic treatment.
• the cells are then inoculated into untreated bacteriological petri dishes for cell culture (Falcon® bacteriological bacterium box, Corning, ref: 351007), previously containing the dermal papillae equivalents, at a density of 6000 cells / cm 2 , in the serum-free nutrient culture medium B as described in Example 1.
• the hair follicles according to the invention thus obtained have the morphological and functional characteristics of a hair follicle in vivo.
• the preparation of the dermal papilla equivalent was carried out according to the preparation protocol described in Example 2 of WO 2009/118283.
• Non-essential amino acid (AANE) (Gibco No. 11140-035)
• Insulin-Transferrin-Sodium Selenite (ITS) (Fisher Scientific No. 10524233)
• Culture support 3D Plate 6 wells ULA (ultra low attachment) flat bottom.
• Cell density 6660 cells / cm 2 .
• the preparation of the hair follicle equivalent was performed according to the preparation protocol described in Example 3 of WO 2009/118283.
• fibroblasts from the dermal papilla 500 000 DP / F75 ie 6660 DP / cm 2 ;
• Example 5 Comparative Exclude Invention: Culture of fibroblasts derived from dermal papilla on a 2D culture medium for cell culture (i.e. support for adhesion of cells).
• Example 6 Comparative Excludes Invention: Dermal papilla obtained in a culture medium containing serum as described in Higgins et al. ⁇ "Modeling the hate follicle dermal papilla using spheroid cell cultures”)
• ALPL alkaline phosphatase
• VCAN versican
• SFRP2 secreted frizzled related protein 2
• IX Material and Method was performed the st step of producing a spheroid comprising fibroblasts from dermal papilla and proliferative epithelial cells (matrix cells) in DMEM + 10% serum in a 96 well plate ULA bottom U.

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