EP3704226A1 - Method for the in vitro preparation of dermal papilla and hair follicle equivalents - Google Patents

Method for the in vitro preparation of dermal papilla and hair follicle equivalents

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Publication number
EP3704226A1
EP3704226A1 EP18803336.9A EP18803336A EP3704226A1 EP 3704226 A1 EP3704226 A1 EP 3704226A1 EP 18803336 A EP18803336 A EP 18803336A EP 3704226 A1 EP3704226 A1 EP 3704226A1
Authority
EP
European Patent Office
Prior art keywords
cells
dermal papilla
equivalent
hair
hair follicle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18803336.9A
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German (de)
French (fr)
Inventor
Khalid Bakkar
Theebah SELLATHURAI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LOreal SA
Original Assignee
LOreal SA
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Filing date
Publication date
Application filed by LOreal SA filed Critical LOreal SA
Publication of EP3704226A1 publication Critical patent/EP3704226A1/en
Pending legal-status Critical Current

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C12N5/0628—Hair stem cells; Hair progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • G—PHYSICS
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    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
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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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Abstract

The invention concerns a method for the in vitro preparation of a dermal papilla equivalent from fibroblasts from the dermal papilla and/or the connective tissue sheath; a method for the in vitro preparation of a hair follicle equivalent by culturing proliferative epithelial cells on said dermal papilla obtained in this way; the in vitro equivalents of dermal papilla and hair follicles produced by the abovementioned methods, the uses thereof for treating alopecia and for evaluating the effect of cosmetic, pharmaceutical or dermatological products.

Description

Procédé de préparation in vitro d'équivalents de papille dermique et de follicule pileux  Process for the in vitro preparation of dermal papilla and hair follicle equivalents
La présente invention concerne un procédé de préparation in vitro d'un équivalent de papille dermique à partir de fïbroblastes issus de la papille dermique et/ou de la gaine conjonctive. La présente invention concerne également un procédé de préparation in vitro d'un équivalent de follicule pileux par mise en culture de cellules épithéliales prolifératives sur lesdites papilles dermiques ainsi obtenues. 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.
Elle concerne, de même, les équivalents in vitro de papilles dermiques et follicules pileux produits par les procédés précités, leurs utilisations pour le traitement de l'alopécie et pour l'évaluation de l'effet de produits cosmétiques, pharmaceutiques ou dermatologiques.  It also relates to the in vitro equivalents of dermal papillae and hair follicles produced by the abovementioned processes, their uses for the treatment of alopecia and for the evaluation of the effect of cosmetic, pharmaceutical or dermatological products.
Durant sa vie, le cheveu connaît trois phases de développement de durées très inégales : During his life, the hair experiences three phases of development of very unequal durations:
La phase anagène est la phase active du cheveu, celle pendant laquelle il vit et pousse régulièrement. Elle dure de 4 à 7 ans. Les cellules germinales qui entourent la papille du bulbe de la racine du cheveu fabriquent continuellement la matière permettant au cheveu de vivre et de pousser. Ensuite, la multiplication des cellules germinales s'arrête au fond du follicule. La phase active du cheveu est alors terminée ; une autre commence, qui est beaucoup plus courte. 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.
La phase catagène : En 15 jours à peine, le bulbe du cheveu disparait (car il n'est plus alimenté à cause de l'interruption des cellules germinatives) et se transforme à une vitesse accélérée. La papille disparaît, c'est-à-dire que le bulbe creux devient plein ; il se kératinise, se durcit et devient corné. Le cheveu est alors mort ; le follicule se resserre pour expulser le cheveu mort. 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.
La phase télogène est la phase qui dure trois mois environ et pendant laquelle le cheveu mort est en attente de tomber. Afin de tomber, le cheveu doit être poussé dehors par le nouveau cheveu qui pousse à son tour dans le même follicule et qui va expulser l'ancien. 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.
La régénération du follicule pileux se fait alors à partir des cellules-souches, appelées cellules germinales situées dans le " bulge ". Regeneration of the hair follicle is then made from stem cells, called germ cells located in the "bulge".
Le " bulge " est formé par une sous-population cellulaire de la gaine épithéliale externe, appelée cellules germinales, localisée au niveau de la portion moyenne du follicule pileux, et plus exactement au niveau de la zone d'insertion du muscle arrecteur du poil. Ces cellules représentent la partie la plus inférieure de la portion permanente du follicule. 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.
Au niveau du " bulge ", les kératinocytes sont relativement indifférenciés, biochimiquement et ultrastructurellement.  At the level of "bulge", keratinocytes are relatively undifferentiated, biochemically and ultrastructurally.
Ce " bulge " est en position stratégique pour interagir durant la phase télogène tardive, avec l'ascendante papille dermique et initier un nouveau follicule en anagène. Les cellules germinales sont donc des cellules essentielles pour le renouvellement du cheveu. Les cellules germinales (cellules germinatives ou cellules pluripotente ou cellules souches) du cheveu télogène sont impliquées dans la sortie de la phase de dormance du follicule pileux et donc la repousse du cheveu.  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.
Le bulbe pilaire est en forme de poire et il est composé : The hair bulb is pear-shaped and consists of:
De la papille qui est un bourgeonnement d'origine dermique, située à la base du follicule. C'est un site très vascularisé qui participe à la nutrition et à la régulation de la croissance du cheveu par sa réserve en facteurs de croissance et protéines de la matrice extracellulaire. Ces informations seront transmises aux cellules matricielles, élaborées dans la matrice, pour permettre leur différenciation (Rees JL. Genetics of hair and skin color).  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).
De la matrice qui est une zone coiffant la papille dermique, constituée d'un amas de cellules matricielles peu différenciées. C'est le siège d'une intense activité mitotique. Les cellules de la matrice, qui sont localisées dans le bulbe pileux et qui forment un petit amas cellulaire autour de la papille dermique, sont majoritairement constituées de précurseurs de kératinocytes qui constituent une assise germinative et qui prolifèrent rapidement pour se différencier en formant la tige pilaire, jouant ainsi un rôle essentiel dans le cycle pilaire. Du début de la phase anagène jusqu'à la fin de celle-ci, ces cellules matricielles vont proliférer jusqu'à la phase catagène puis disparaître en phase télogène. (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). La différenciation cellulaire va permettre la formation des différents types cellulaires de la gaine épithéliale externe (ORS), interne (1RS) puis de la tige pilaire. C'est aussi cette matrice qui conditionne la forme du cheveu. La matrice se répartit de façon homogène autour d'un axe de symétrie pour le cheveu droit alors qu'elle sera plus importante d'un côté pour le cheveu frisé (Melissopoulos A et Levacher C. Les annexes cutanées. Dans : La peau : structure et physiologie, édition Lavoisier ; 1998. P.57-99). La matrice comprend également des mélanocytes folliculaires qui sont responsables de la pigmentation du cheveu. La prolifération et la différenciation de ces cellules de la matrice sont contrôlées par la papille dermique (Botchkarev VA, ishimoto J. Molecular control of epithelial-mesenchymal interactions during hair follicle cycling. J Invest Dermatol Symp Proc. 2003 Jun;8(l):46-55. Review). Of 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. (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). The cell differentiation will allow the formation of different cell types of the outer epithelial sheath (ORS), internal (1RS) and then the hair shaft. It is also this matrix that conditions the shape of the hair. The matrix is homogeneously distributed around an axis of symmetry for the right hair whereas it will be more important on one side for the curly hair (Melissopoulos A and Levacher C. Appendices skin. In: The Skin: Structure and Physiology, Lavoisier Edition; 1998. P.57-99). 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).
Des gaines épithéliales externe et interne qui sont produites par la matrice supérieure du bulbe pileux aussi appelé zone de kératinisation. La gaine épithéliale externe constitue l'enveloppe externe du follicule : il s'agit d'une invagination de l'épidémie. Celle-ci héberge notamment des cellules souches à partir desquelles le follicule pileux sera cycliquement régénéré. La gaine épithéliale interne sépare la gaine épithéliale externe de la tige pilaire. Cette gaine est constituée de trois types cellulaires organisés en couche concentriques kératinisées qui accompagnent la croissance du cheveu. On distingue la couche de Henlé, la couche de Huxley et la cuticule qui est formée de cellules aplaties dirigé vers la matrice pilaire. External and internal epithelial sheaths that are produced by the upper matrix of the hair bulb also called keratinization zone. 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.
De la tige pilaire qui est en partie visible, c'est le cheveu. La structure de la tige pilaire se compose de trois couches distinctes de l'extérieur vers l'intérieur. On retrouve la cuticule, le cortex et la médulla, toutes composées de cellules kératinisées. 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.
L'alopécie est conditionnée par divers facteurs : génétiques, hormonaux et environnementaux, par le régime alimentaire et l'activité physique. Les cheveux tiennent un rôle esthétique et identitaire essentiel. Alopecia is conditioned by various factors: genetic, hormonal and environmental, diet and physical activity. Hair has an essential aesthetic and identity role.
Ainsi des cheveux sains, vigoureux et une chevelure dense tout au long de la vie est recherché par la plupart des femmes et des hommes.  Healthy, vigorous hair and dense, lifelike hair is sought after by most women and men.
De nombreuses techniques sont connues pour le traitement de l'alopécie telles que la thérapie cellulaire, le traitement au laser ou encore les implants sans chirurgie. Cette dernière apporte un résultat immédiat et se trouve être beaucoup moins invasive que la chirurgie. Many techniques are known for the treatment of alopecia such as cell therapy, laser treatment or implants without surgery. The latter brings an immediate result and is much less invasive than surgery.
Afin d'obtenir des implants, les follicules pileux humains sont obtenus par la culture de différents types cellulaires présents au niveau du bulbe pilaire. Depuis plusieurs années, l'homme du métier a mis en culture in vitro 2D ou 3D des cellules du follicule pileux issues des différents compartiments qui composent le bulbe. In order to obtain implants, human hair follicles are obtained by culturing different types of cells present in the hair bulb. For several years, the skilled person has cultured in vitro 2D or 3D hair follicle cells from different compartments that make up the bulb.
C. Higgins a montré qu'en culture in vitro 2D les cellules de la papille dermique perdent leur capacité d'induction dès les premières mises en culture et amplification. Les sphéroïdes obtenus en culture 2D et 3D décrits par Higgins ne présentent pas les caractéristiques fonctionnelles d'une papille dermique in vivo, en particulier ces sphéroïdes présentent une faible activité à la phosphatase alcaline, au Versican, et SFRP2 (secreted frizzled related protein 2) comme le montre l'exemple 6 ci-après. C. 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.
D'autres équipes comme Park en Corée utilisent un milieu riche en acides aminés et vitamines pour former des DPLTs (dermal papilla like tissue). En particulier, sont décrits respectivement dans les documents suivants WO2009/014272 et US2008/0145929 des équivalents in vitro de papilles dermiques préparés à partir de cellules souches mésenchymateuses issues de cordon ombilical et de fîbroblastes issus de papilles dermiques. Cependant, l'utilisation de cellules souches mésenchymateuses provenant d'un échantillon biologique de cordon ombilical décrite dans le document WO2009/014272 et l'utilisation d'un support de culture 2D traité pour la culture cellulaire n'empêchant pas l'adhésion des cellules décrite dans le document US2008/0145929, ne permettent pas d'obtenir des équivalents in vitro de papilles dermiques présentant les caractéristiques essentielles d'une papille dermique vivo, en particulier d'un point de vue morphologique et/ou fonctionnel et notamment une activité enzymatique phosphatase alcaline positive. Other teams like Park in Korea use a medium rich in amino acids and vitamins to form DPLTs (dermal papilla like tissue). In particular, the following documents WO2009 / 014272 and US2008 / 0145929 respectively describe the in vitro equivalents of dermal papillae prepared from mesenchymal stem cells derived from umbilical cord and from fibroblasts derived from dermal papillae. However, the use of mesenchymal stem cells from an umbilical cord biological sample described in WO2009 / 014272 and the use of a 2D culture medium treated for cell culture does not prevent cell adhesion. described in document US2008 / 0145929, do not make it possible to obtain in vitro equivalents of dermal papillae having the essential characteristics of an in vivo dermal papilla, in particular from a morphological and / or functional point of view and in particular an enzymatic activity. positive alkaline phosphatase.
Par ailleurs, aucun des modèles précités ne contient de cellules épithéliales prolifératives comme les cellules de la matrice ou les cellules germinales, dont la présence et la quantité sont essentielles à la formation d'une structure folliculaire. Moreover, 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.
Le document WO2017/055358 divulgue la capacité des cellules de la matrice à régénérer un micro follicule pileux en présence d'inhibiteur de ROCK. Néanmoins ces cellules de la matrice sont ensemencées sur des fîbroblastes ayant perdu leur capacité à proliférer (fîbroblastes 3T3, fîbroblastes humains irradiés ou traités à la mitomycine), et non sur des papilles dermiques. Ceci implique ainsi des différences fonctionnelles et morphologiques comme le montre la figure 10 ci-après. Il existe donc un besoin de pouvoir disposer d'équivalents in vitro de papilles dermique et de follicule pileux présentant respectivement la plupart des caractéristiques fonctionnelles et morphologiques d'une papille dermique et d'un follicule pileux vivo, de préférence un follicule pileux humain et notamment susceptible de se régénérer. 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. There is therefore a need to be able to have in vitro equivalents of dermal papillae and hair follicles respectively presenting most of the functional and morphological characteristics of a dermal papilla and a hair follicle in vivo, preferably a human hair follicle, and in particular likely to regenerate.
De façon surprenante, la demanderesse a mis en évidence d'une part que la culture de fïbroblastes issus de la papille dermique et/ou de la gaine conjonctive dans un milieu exempt de sérum sur des supports de culture 2D ou 3D particuliers de type microplaques fond rond, ces supports ne permettant pas l'adhésion des cellules, permet d'obtenir un équivalent in vitro de papille dermique présentant la plupart des caractéristiques fonctionnelles et morphologiques d'une papille dermique vivo. Surprisingly, 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.
D'autre part que l'ensemencement de cellules épithéliales prolifératives telles que les cellules de la matrice et/ou les cellules germinales sur lesdits équivalents in vitro de papilles dermiques obtenus permet d'obtenir un équivalent in vitro de follicule pileux présentant la plupart des caractéristiques fonctionnelles et morphologiques d'un follicule vivo humain, et notamment susceptible de se régénérer.  On the other hand, 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.
Par conséquent, de tels équivalents de papilles dermique et de follicule pileux s'avèrent être particulièrement intéressants pour l'étude de la morphogénèse d'un follicule pileux, pour les tests prédictifs de l'activité d'actifs cosmétiques et/ou pharmaceutiques ainsi que pour le traitement prophylactique ou thérapeutique d'un état de pilosité réduite, et le traitement de l'alopécie. Therefore, such equivalents of dermal papillae and hair follicle prove to be particularly interesting for the study of the morphogenesis of a hair follicle, for the predictive tests of the activity of cosmetic and / or pharmaceutical active agents as well as for the prophylactic or therapeutic treatment of a state of reduced hairiness, and the treatment of alopecia.
Ainsi, selon un premier de ses objets, la présente invention concerne un procédé de préparation in vitro d'un équivalent de papille dermique comprenant au moins une étape de mise en culture de fïbroblastes issus de la papille dermique et/ou de la gaine conjonctive sur un support comprenant un milieu de culture nutritif B exempt de sérum pendant une période de temps suffisante pour permettre auxdits fïbroblastes de se détacher dudit support et de se regrouper pour former au moins un sphéroïde ; la surface dudit support utilisé ne permettant pas l'adhésion des cellules ; ledit support de culture est choisi parmi les supports de culture 2D ou 3D microplaque fond rond. Thus, according to a first of its objects, 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.
Un second objet de la présente invention se rapporte à un équivalent in vitro de papille dermique susceptible d'être obtenu selon le procédé précité. Un troisième objet de la présente invention se rapporte à l'utilisation d'un équivalent de papille dermique selon l'invention et de cellules épithéliales prolifératives telles que les cellules de la matrice et/ou les cellules germinales pour la préparation d'un équivalent in vitro de follicule pileux. 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.
Selon un autre de ses objets l'invention concerne un procédé de préparation in vitro d'un équivalent de follicule pileux comprenant au moins une étape de culture de cellules épithéliales prolifératives en présence d'au moins un équivalent de papille dermique selon l'invention pendant une période de temps suffisante pour permettre une différenciation desdites cellules épithéliales prolifératives en kératinocytes positifs pour les marqueurs K85 et K35. According to another of its objects 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.
La présente invention se rapporte encore à un équivalent in vitro de follicule pileux susceptible d'être obtenu selon le procédé précité. La présente invention concerne également les utilisations thérapeutiques de l'équivalent de follicule pileux selon l'invention dans le traitement prophylactique ou thérapeutique d'un état de pilosité réduite, et le traitement de l'alopécie. 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.
Selon un autre de ses objets l'invention concerne l'utilisation de l'équivalent de follicule pileux selon l'invention pour le test in vitro d'ef ets d'actifs sur les propriétés pileuses, et en particulier pour l'identification de composé modulateur de la pousse des poils et/ou des cheveux. According to another of its objects 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.
La présente invention se rapporte aussi à un procédé de criblage de composés modulateurs de la pousse des poils et/ou des cheveux mettant en œuvre l'équivalent de follicule pileux selon l'invention. 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.
Description détaillée de l'invention Detailed description of the invention
Fibroblastes fibroblasts
Au sens de la présente invention, on entend par « fibroblastes issus de la papille dermique » des fibroblastes prélevés à partir d'une microdissection d'un follicule pileux en phase anagène au niveau du bourgeonnement d'origine dermique situé à la base du follicule pileux. Egalement, on entend par « fibroblastes issus de la gaine conjonctive » des fïbroblastes prélevés à partir d'une microdissection d'un follicule pileux en phase anagène à la base du follicule pileux sous la papille dermique. Les fïbroblastes issus de la papille dermique ou de la gaine conjonctive sont de préférence prélevés à partir de déchets opératoires ou biopsies comme par exemple des déchets de lifting ou un prélèvement de scalp car un simple arrachage du cheveu ne permet pas de récupérer ces cellules. En particulier, les fïbroblastes utilisés dans le cadre de la présente invention ne sont pas des fïbroblastes dont la prolifération a été préalablement stoppée, préférentiellement en les ayant préalablement irradiés (par exemple, aux rayons gamma) ou préalablement traités à la mitomycine. De préférence, les fïbroblastes selon l'invention ne sont pas des fibroblastes 3T3. Cellules épithéliales prolifératives For the purposes of the present invention, the term "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 . Also, "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. In particular, 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. Preferably, the fibroblasts according to the invention are not 3T3 fibroblasts. Proliferative epithelial cells
On entend par « cellules épithéliales prolifératives » des cellules épithéliales présentent au niveau du follicule pileux et capables de donner naissance à tous les types cellulaires présents dans un cheveu. Les cellules épithéliales prolifératives sont de préférence choisies parmi les cellules de la matrice et/ou les cellules germinales.  The term "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.
On entend par « cellules de la matrice », les cellules localisées dans le bulbe pileux et qui forment un petit amas cellulaire autour de la papille dermique (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). Ces cellules pourront être échantillonnées, amplifiées et conservées en banques de tissus en vue d'une utilisation ultérieure. The term "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.
Pour préserver l'intégrité du tissu matriciel, ces cellules pourront être prélevées selon le procédé qui suit : des follicules pileux sont placés dans une boite de Pétri contenant un milieu de culture minimum additionné de 2% d'antibiotique et des acides aminés non essentiels.  To preserve the integrity of the matrix tissue, 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.
La région bulbaire est coupée au sommet de la papille dermique ainsi l'épithélium du bulbe est séparé de la papille dermique et de la gaine conjonctive. 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.
Au sens de l'invention, on entend par « cellules germinales », les cellules souches présentes au niveau du « bulge ». De préférence, les cellules germinales sont prélevées en phase télogène. Au sens de l'invention on entend par « télogène » le follicule qui contenait le cheveu en phase télogène. For the purposes of the invention, the term "germinal cells" means the stem cells present at the level of the "bulge". Preferably, the germinal cells are removed in the telogen phase. For the purposes of the invention, "telogen" is understood to mean the follicle which contained the hair in the telogen phase.
Les cellules germinales pourront être prélevées selon le procédé qui suit : des follicules pileux en phase télogène sont placés dans une boite de Pétri contenant un milieu de culture minimum additionné de 2% d'antibiotique et des acides aminés non essentiels. 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.
La région des cellules germinales, appelée le bulge, est extraite de la gaine conjonctive et ensuite placée dans une boite de Pétri contenant une couche nourricière de fïbroblastes 3T3i. Cette technique de microdissection permet ainsi de préserver la quantité et l'intégrité des cellules, car elles ne sont pas séparées les unes des autres.  The germ cell region, called the bulge, 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.
Procédé de préparation d'un équivalent de papille dermique La présente invention concerne un procédé de préparation in vitro d'un équivalent de papille dermique comprenant au moins une étape de mise en culture de fïbroblastes issus de la papille dermique et/ou de la gaine conjonctive sur un support comprenant un milieu de culture nutritif B exempt de sérum pendant une période de temps suffisante pour permettre auxdits fïbroblastes de se détacher dudit support et de se regrouper pour former au moins un sphéroïde ; la surface dudit support utilisé ne permettant pas l'adhésion des cellules; ledit support de culture est choisi parmi les supports de culture 2D ou 3D microplaque fond rond. On entend par « sphéroïde » un microtissu 3D sous la forme d'une sphère dans lequel les cellules vont s'organiser et synthétiser de la matrice extracellulaire. On considère que l'on obtient ledit équivalent de papille dermique lorsque l'on voit apparaître après mise en œuvre du procédé selon l'invention au moins un sphéroïde. Au sens de la présente invention, on entend par « exempt de sérum », un milieu de culture comprenant moins de 5% en volume de sérum, moins de 4% en volume, moins de 3% en volume, moins de 2% en volume, moins de 1% en volume, de préférence 0% en volume de sérum par rapport au volume total du milieu de culture. 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. The term "spheroid" means a 3D microtissu in the form of a sphere in which the cells will be organized and synthesize the extracellular matrix. It is considered that one obtains said equivalent of dermal papilla when one sees appear after implementation of the method according to the invention at least one spheroid. For the purposes of the present invention, 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.
On entend par « sérum », les protéines sériques contenues dans le sérum, et en particulier au moins une protéine sérique choisie parmi les albumines, les globulines telles que les alpha 1- globulines, les alpha 2-globulines, les bêtaglobulines, les gammaglobulines, et leurs mélanges. Lors de ladite étape de mise en culture des fïbroblastes issus de la papille dermique et/ou de la gaine conjonctive, on observe la formation de clusters à au moins 1 jour, puis lesdits clusters forment des agrégats à au moins 2 jours. On observe enfin l'apparition de sphéroïdes à au moins 3 jours. "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. During said step of culturing fibroblasts derived from the dermal papilla and / or the conjunctive sheath, the formation of clusters at least 1 day is observed, then said clusters form aggregates at least 2 days. We finally observe the appearance of spheroids at least 3 days.
On entend par « clusters » au sens de la présente invention un regroupement de cellules en 2D. Au sens de la présente invention on entend par « agrégats » ou « sphéroïde précoce » un amas de cellules en 3D de forme irrégulière, non organisé et ne synthétisant pas encore de matrice extracellulaire. Dans un mode de réalisation préféré ledit milieu de culture nutritif B comprend de 500 à 1500 mg/L d'acides aminés, de 2 à 18 mg/L de vitamines, de 1500 à 4500 mg/L de glucose, de 8750 à 10000 mg/L de sels inorganiques, de 2 à 20 μg/ml d'insuline, de 2 à 60 ng/ml d'hydrocortisone, et éventuellement de 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotiques. Avantageusement, ledit milieu de culture nutritif B comprend de 600 à 1250 mg/L d'acides aminés, de 5 à 15 mg/L de vitamines, de 1750 à 2250 mg/L de glucose, de 9000 à 9750 mg/L de sels inorganiques, de 5 à 15 μg/ml d'insuline, de 5 à 50 ng/ml d'hydrocortisone, et éventuellement de 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotique. Encore mieux, ledit milieu de culture nutritif B comprend de 700 à 1150 mg/L d'acides aminés, de 5 à 15 mg/L de vitamines, de 1750 à 2250 mg/L de glucose, de 9000 à 9750 mg/L de sels inorganiques, de 5 à 15 μg/ml d'insuline, de 5 à 50 ng/ml d'hydrocortisone, et éventuellement de 100 à 180 μg/ml d'antibiotiques et/ou d'antimycotique. Les acides aminés présents dans ledit milieu B sont de préférence choisis parmi la glycine, L- glutamine, L-alanine, L-arginine, L-asparagine-H20, L-acide aspartique, L-cystéine, L-cystine 2HC1, L-acide glutamique, L-histidine, L-isoleucine, L-leucine, chlorhydrate de L-lysine, L- méthionine, L-phénylalanine, L-proline, L-sérine, L-thréonine, L-tryptophane, sel disodique dihydraté de L-tyrosine, L-valine, et leurs mélanges. For the purposes of the present invention, the term "clusters" refers to a grouping of cells in 2D. For the purposes of the present invention, the term "aggregates" or "early spheroid" means a cluster of 3D cells of irregular shape, unorganized and not yet synthesizing extracellular matrix. In a preferred embodiment 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 μg / ml of insulin, from 2 to 60 ng / ml of hydrocortisone, and optionally from 50 to 200 μg / ml of antibiotics and / or antimycotics. Advantageously, 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. inorganic, from 5 to 15 μg / ml of insulin, from 5 to 50 ng / ml of hydrocortisone, and optionally from 50 to 200 μg / ml of antibiotics and / or antimycotic. Even better, 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 μg / ml of insulin, from 5 to 50 ng / ml of hydrocortisone, and optionally from 100 to 180 μg / 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.
Les vitamines présentes dans ledit milieu B sont de préférence choisies parmi l'acide ascorbique, biotine, chlorure de choline, D-calcium pantothénate, ergocalciferol, acide folique, menadione sodium bisulfate, niacinamide, chlorhydrate de pyridoxal, riboflavine, chlorhydrate de thiamine, rétinyl acétate, vitamine B12, alpha tocopherol phosphate disodique, i-inositol, et leurs mélanges. 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.
Les sels inorganiques présents dans ledit milieu B sont de préférence choisis parmi le chlorure de calcium anhydre (CaC12), sulfate de cuivre pentahydraté (CuS04-5H20), sulfate ferreux heptahydraté (FeS04-7H20), sulfate de magnésium anhydre (MgS04), sulfate de manganèse monohydraté (MnSO4-H20), chlorure de potassium (KO), bicarbonate de sodium (NaHC03), chlorure de sodium (NaCl), dihydrogénophosphate de sodium anhydre (NaH2P04), sulfate de zinc heptahydraté (ZnS04-7H20), et leurs mélanges. 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.
A titre d'exemples d'antibiotiques présents dans ledit milieu B on peut citer la pénicilline, la streptomycine, et leurs mélanges. Examples of antibiotics present in said medium B include penicillin, streptomycin, and mixtures thereof.
A titre d'exemples d'antimycotiques présents dans ledit milieu B on peut citer notamment l'amphotéricine B.  Examples of antimycotics present in said medium B include amphotericin B.
Ledit milieu de culture nutritif B exempt de sérum utilisé comprend de préférence moins de 15 μg/ml de facteurs de croissance, de préférence moins de 12 μg/ml de facteurs de croissance. En particulier, les facteurs de croissance présents dans ledit milieu nutritif B sont choisis parmi l'insuline et/ou l'hydrocortisone. Said serum-free nutrient culture medium B preferably comprises less than 15 μg / ml of growth factors, preferably less than 12 μg / ml of growth factors. In particular, the growth factors present in said nutrient medium B are selected from insulin and / or hydrocortisone.
Dans un mode de réalisation particulièrement préféré, ledit milieu de culture nutritif B comprend de 80% à 99% en volume de milieu de Williams E, de 250 à 350 mg/L de glutamine, de 2 à 20 μg/ml d'insuline, de 2 à 60 ng/ml d'hydrocortisone, et éventuellement de 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotique. Encore mieux, ledit milieu de culture nutritif B comprend de 95% à 98% en volume de milieu de Williams E, de 280 à 320 mg/L de glutamine, de 5 à 15 μg/ml d'insuline, de 5 à 50 ng/ml d'hydrocortisone, et éventuellement de 100 à 180 μg/ml d'antibiotiques et/ou d'antimycotique. In a particularly preferred embodiment, 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 μg / ml of insulin, from 2 to 60 ng / ml of hydrocortisone, and optionally from 50 to 200 μg / ml of antibiotics and / or antimycotic. Even better, 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 μg / ml of insulin, from 5 to 50 ng / ml of hydrocortisone, and optionally from 100 to 180 μg / ml of antibiotics and / or antimycotic.
Le milieu de Williams E est en particulier exempt de glutamine. Les fïbroblastes sont de préférence cultivés pendant au moins 3 jours, encore mieux entre 4 et 21 jours. 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.
Les fïbroblastes sont de préférence ensemencés à forte densité. En particulier, en une densité d'au moins 3000 cellules/cm2, de préférence d'au moins 9000 cellules/cm2, encore mieux d'au moins 14000 cellules/cm2, de manière encore plus préférée en une densité comprise entre 14000 cellules/cm2 et 47600 cellules/cm2. The fibroblasts are preferably seeded at high density. In particular, in 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 .
Dans un mode de réalisation préféré, les fïbroblastes sont ensemencés en une densité comprise entre 23500 cellules/cm2 et 24500 cellules/cm2, encore mieux en une densité de 23800 cellules/cm2. In a preferred embodiment, 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 .
Le support ne permettant pas l'adhésion des cellules, en particulier des fïbroblastes, est choisi parmi les supports de culture 2D et 3D microplaque fond rond. The support that does not allow the adhesion of cells, in particular fibroblasts, is chosen from 2D and 3D microplate round-bottom culture media.
Avantageusement, les supports 2D et 3D utilisés dans le cadre de la présente invention ne sont pas recouverts de collagène. Advantageously, the 2D and 3D supports used in the context of the present invention are not coated with collagen.
Parmi les supports de culture 2D, on peut citer notamment les boîtes de pétri bactériologiques non traitées pour la culture cellulaire, en particulier en plastique, ne permettant pas l'adhésion des cellules. Among the 2D culture media, there may be mentioned bacteriological petri dishes not treated for cell culture, in particular plastic, not allowing the adhesion of cells.
La surface dudit support est de préférence neutre. On entend par « surface neutre » au sens de la présente invention, une surface non chargée.  The surface of said support is preferably neutral. The term "neutral surface" in the sense of the present invention, an uncharged surface.
La surface dudit support peut être hydrophobe ou hydrophile, de préférence hydrophobe. Lorsque ladite surface est hydrophile, celle-ci est recouverte d'une substance telle qu'elle empêche toute adhésion cellulaire, en particulier, une telle substance peut être choisie parmi les hydrogels liés de manière covalente à la surface dudit support.  The surface of said support may be hydrophobic or hydrophilic, preferably hydrophobic. When said surface is 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.
Dans un mode particulièrement préféré la surface dudit support est neutre et hydrophobe. On pourra utiliser à titre de support de culture 2D, une boite de pétri bactériologique commercialisée sous le nom de Falcon® commercialisé par la société Corning (référence : 351007).  In a particularly preferred embodiment the surface of said support is neutral and hydrophobic. As a 2D culture support, a bacteriological petri dish sold under the name Falcon® sold by the company Corning (reference: 351007) may be used.
Parmi les supports de culture 3D microplaque fond rond, on pourra notamment utiliser une microplaque 96 puits à fond rond commercialisée sous le nom de costar® par la société Corning (référence : 7007). Among the 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.
De préférence, le support de culture 3D n'est pas une microplaque à fond plat. Preferably, the 3D culture support is not a flat-bottomed microplate.
Dans un premier mode de réalisation, lesdits fïbroblastes issus de la papille dermique et/ou de la gaine sont ensemencés sur un support de culture 2D ne permettant pas l'adhésion des cellules en une densité d'au moins 14000 cellules/cm2, de préférence en une densité comprise entre 14000 cellules/cm2 et 47600 cellules/cm2, encore mieux entre 23500 cellules/cm2 et 24500 cellules/cm2. In a first embodiment, 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 .
Dans un second mode de réalisation préféré, lesdits fïbroblastes issus de la papille dermique et/ou de la gaine sont ensemencés sur un support de culture 3D microplaque à fond rond ne permettant pas l'adhésion des cellules en une densité d'au moins 3000 cellules/cm2, de préférence d'au moins 9000 cellules/cm2, encore mieux en une densité comprise entre 9000 cellules/cm2 à 20 000 cellules/cm2- Le procédé de préparation in vitro d'un équivalent de papille dermique selon l'invention peut comprendre en outre une étape préliminaire d'amplification desdits fïbroblastes issus de la papille dermique et/ou de fïbroblastes issus de la gaine conjonctive dans un milieu de culture nutritif A ; ledit milieu de culture nutritif A comprenant au moins du sérum, en particulier du sérum de veau fœtal. In a second preferred embodiment, 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. / cm 2 , preferably at least 9000 cells / cm 2 , more preferably in a density of between 9000 cells / cm 2 to 20 000 cells / cm 2 - 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.
De préférence, le support de culture utilisé pour cette étape d'amplification est un support traité pour permettre l'adhésion des cellules. Ces supports sont ceux classiquement utilisés pour la culture cellulaire et sont donc bien connus de l'homme du métier. De préférence, ledit milieu de culture nutritif A utilisé à ladite étape d'amplification comprend de 500 à 2000 mg/L d'acides aminés, de 15 à 35 mg/L de vitamines, de 2500 à 4500 mg/L de glucose, de 7500 à 9500 mg/L de sels inorganiques, de 5% à 30% en volume de sérum de veau fœtal, et éventuellement de 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotiques. Préférentiellement, ledit milieu de culture nutritif A utilisé à ladite étape d'amplification comprend de 750 à 1800 mg/L d'acides aminés, de 20 à 30 mg/L de vitamines, de 3000 à 4000 mg/L de glucose, de 8000 à 9000 mg/L de sels inorganiques, de 10% à 25% en volume de sérum de veau fœtal, et éventuellement de 100 à 180 μg/ml d'antibiotiques et/ou d'antimycotique. Les acides aminés présents dans ledit milieu de culture A utilisé à ladite étape d'amplification sont de préférence choisis parmi la glycine, L-alanyl-glutamine, chlorhydrate de L-arginine, L- cystine 2HC1, L-histidine hydrochloride-H20, L-isoleucine, L-leucine, chlorhydrate de L- lysine, L-méthionine, L-phénylalanine, L-serine, L-thréonine, L-tryptophane, L-tyrosine, L- valine, L-alanine, L-asparagine, L-acide aspartique, L-acide glutamique, L-proline, et leurs mélanges. Preferably, 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. Preferably, 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 μg / ml of antibiotics and / or antimycotics. Preferably, 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 μg / 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.
Les vitamines présentes dans ledit milieu de culture A utilisé à ladite étape d'amplification sont de préférence choisies parmi la chlorure de choline, D-calcium pantothenate, l'acide folique, niacinamide, chlorhydrate de pyridoxine, riboflavine, chlorhydrate de thiamine, i-inositol et leurs mélanges. 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.
Les sels inorganiques présents dans ledit milieu de culture A utilisé à ladite étape d'amplification sont de préférence choisis parmi le chlorure de calcium (CaCl2), le sulfate de magnésium (MgSC^), le nitrate de fer (Fe(N03), 9H20), le chlorure de potassium (KCl), le bicarbonate de sodium (NaHC03), le chlorure de sodium (NaCl), le dihydrogénophosphate de sodium (NaH2P04, 4H20), et leurs mélanges. A titre d'exemples d'antibiotiques présents dans ledit milieu A on peut citer la pénicilline, la streptomycine, et leurs mélanges. 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. Examples of antibiotics present in said medium A include penicillin, streptomycin, and mixtures thereof.
A titre d'exemples d'antimycotiques présents dans ledit milieu A on peut citer notamment l'amphotéricine B. Dans un mode de réalisation particulièrement préféré ledit milieu de culture nutritif A utilisé à ladite étape d'amplification comprend de 70% à 80% en volume de milieu DMEM Glutamax, de 5% à 25% de sérum de veau fœtal, de 50 à 90 mg/L d'acides aminés non essentiels, et éventuellement de 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotiques. Encore mieux, ledit milieu de culture nutritif A comprend 78% en volume de milieu DMEM Glutamax, 20% de sérum de veau fœtal, de 60 à 80 mg/L d'acides aminés non essentiels, et éventuellement de 100 à 180 μg/ml d'antibiotiques et/ou d'antimycotiques.  Examples of antimycotics present in said medium A include amphotericin B. In a particularly preferred embodiment, 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 μg / 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 μg / ml antibiotics and / or antimycotics.
Les acides aminés non essentiels présents dans ledit milieu de culture A utilisé à ladite étape d'amplification sont choisis parmi la L-alanine, L-asparagine, L-acide aspartique, L-acide glutamique, L-proline, L-sérine, et leurs mélanges. 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.
Dans un mode de réalisation particulièrement préféré, le procédé de préparation d'un équivalent in vitro de papille dermique selon l'invention comprend en outre préalablement à l'étape de mise en culture desdits fîbroblastes, les étapes préliminaires suivantes : a, isoler un follicule pileux en phase anagène à partir d'un prélèvement de scalp ; b récupérer les fîbroblastes de la papille dermique et/ou de la gaine conjonctive à l'aide d'une microdissection de la papille dermique et/ou de la gaine conjonctive ; In a particularly preferred embodiment, 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;
c effectuer une amplification desdits fîbroblastes de la papille dermique et/ou de fîbroblastes de la gaine conjonctive dans un milieu de culture nutritif A constitué de DMEM Glutamax supplémenté de 20% en volume de sérum fœtal de veau (SVF), 50 à 90 mg/L d'acides aminés non essentiels, et éventuellement 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotique.  c amplification of said dermal papilla fibroblasts and / or fibroblasts of the conjunctive sheath in a nutrient culture medium A consisting of DMEM Glutamax supplemented with 20% by volume of fetal calf serum (FCS), 50 to 90 mg / ml. L of non-essential amino acids, and possibly 50 to 200 μg / ml of antibiotics and / or antimycotic.
La présente invention concerne également un équivalent in vitro de papille dermique susceptible d'être obtenu par le procédé selon l'invention tel que décrit ci-dessus. The present invention also relates to an in vitro equivalent dermal papilla obtainable by the method according to the invention as described above.
De préférence, l'équivalent de papille dermique selon l'invention est caractérisé en ce qu'il présente une activité phosphatase alcaline positive, et éventuellement une expression positive des protéines choisies parmi BMP2 (Bone morphogenetic protein 2), SFRP2 (secreted frizzled related protein 2), CORIN, SOX2, VCAN (Versican) et/ou PLC (Perlecan). Preferably, 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).
L'équivalent de papille dermique selon l'invention est de préférence caractérisé en ce qu'il est constitué de cellules issues d'une étape de mise en culture de fîbroblastes issus de la papille dermique et/ou de la gaine conjonctive sur un support comprenant un milieu de culture nutritif B exempt de sérum pendant une période de temps suffisante pour permettre auxdits fîbroblastes de se détacher dudit support et de se regrouper pour former au moins un sphéroïde ; ledit support ne permettant pas l'adhésion des cellules ; ledit support de culture est choisi parmi les supports de culture 2D ou 3D microplaque fond rond. L'équivalent de papille dermique selon l'invention est en particulier une sphère de diamètre allant de ΙΟΟμιη à 250μιη. De préférence, l'équivalent de papille dermique selon l'invention est une sphère d'environ 200μιη de diamètre. 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 ΙΟΟμιη to 250μιη. Preferably, the dermal papilla equivalent according to the invention is a sphere of approximately 200 μm in diameter.
L'activité enzymatique de la phosphatase alcaline peut par exemple être mesurée par le kit phosphatase alcaline NBT/BCIP commercialisé par le Laboratoire Roche (réf : 11 681 451 001 au 30 octobre 2017) où le BCIP (sel de 5-Bromo-4-chloro-3-indolyl phosphate toluidine), substrat de la phosphatase alcaline, sera d'abord déphosphorylé puis oxydé pour donner un produit coloré bleu. L'expression protéique des marqueurs BMP2, SFRP2, CORIN, SOX2, VCAN et PLC peut par exemple être mesurée à l'aide de la technique d'immunofluorescence, technique par ailleurs bien connue de l'homme du métier. Procédé de préparation d'un équivalent de follicule pileuxThe 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
La présente invention concerne en outre l'utilisation d'un équivalent in vitro de papille dermique selon l'invention et de cellules épithéliales prolifératives pour la préparation d'un équivalent in vitro de follicule pileux. La présente invention concerne également un procédé de préparation in vitro d'un équivalent de follicule pileux comprenant au moins une étape de culture de cellules épithéliales prolifératives en présence d'au moins un équivalent de papille dermique selon l'invention pendant une période de temps suffisante pour permettre une différenciation desdites cellules épithéliales prolifératives en kératinocytes positifs pour les marqueurs K85 et K35. 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.
Les cellules épithéliales prolifératives sont de préférence choisies parmi les cellules de la matrice, les cellules germinales telles que définies précédemment, et leurs mélanges. The proliferative epithelial cells are preferably chosen from the cells of the matrix, the germinal cells as defined above, and their mixtures.
Dans un mode de réalisation particulièrement préféré, les cellules épithéliales prolifératives utilisées dans le cadre de la présente invention sont les cellules de la matrice. In a particularly preferred embodiment, the proliferative epithelial cells used in the context of the present invention are the cells of the matrix.
Etape de culture des cellules épithéliales prolifératives en présence d'au moins un équivalent in vitro de papille dermique Proliferative epithelial cell culture step in the presence of at least one in vitro dermal papilla equivalent
Les cellules épithéliales prolifératives ensemencées sur au moins un équivalent de papille dermique selon l'invention sont cultivées à 37°C et à 5% de C02, dans un milieu défini, comme celui décrit par Philpott (1993).  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).
En particulier, les cellules épithéliales prolifératives sont cultivées dans le même milieu de culture nutritif que celui utilisé pour la préparation des équivalents de papille dermique selon l'invention, en particulier le milieu de culture nutritif B exempt de sérum. In particular, 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.
Dans un mode de réalisation particulièrement préféré, ledit équivalent de papille dermique est obtenu par le procédé de préparation d'équivalent de papille dermique tel que précité au paragraphe « Procédé de préparation d'un équivalent de papille dermique ». L'étape de culture desdites cellules épithéliales prolifératives en présence d'au moins un équivalent de papille dermique selon l'invention est réalisée de préférence sur les supports 2D ou 3D tels que définis précédemment dont la surface ne permet pas l'adhésion de cellules. Les cellules épithéliales prolifératives sont de préférence ensemencées à forte densité. De préférence, les cellules épithéliales prolifératives sont ensemencées en une densité d'au moins 2000 cellules/cm2, de préférence d'au moins 6000 cellules/cm2, et encore mieux en une densité comprise entre 6000 et 10000 cellules/cm2- De préférence, lesdites cellules épithéliales prolifératives sont cultivées pendant une période de temps suffisante pour permettre la formation d'au moins une structure tubulaire, encore appelée organoïde folliculaire. In a particularly preferred embodiment, 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. Preferably, 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 . Preferably, 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.
Au sens de l'invention on entend par « structure tubulaire » ou « organoïde folliculaire », le bourgeonnement observé après culture d'au moins un équivalent de papille dermique selon l'invention associé à des cellules épithéliales prolifératives choisies parmi les cellules de la matrice et/ou les cellules germinales. For the purposes of the invention, the term "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.
Lesdites cellules épithéliales prolifératives sont cultivées en présence d'un équivalent de papille dermique pendant au moins 3 jours, de préférence pendant au moins 5 jours, encore mieux entre 5 et 20 jours, préférentiellement entre 5 et 15 jours. 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.
Dans un mode de réalisation particulier, le procédé de préparation de l'équivalent de follicule pileux selon l'invention comprend un étape d'ajout dans le milieu de culture nutritif B de facteurs de croissance choisi parmi les protéines de la famille WNT, en particulier la protéine WNT3A, les protéines de la famille BMP, en particulier les protéines BMP2 et BMP4, et leurs mélanges. L'ajout desdits facteurs de croissance est réalisé entre le 1er jour et le 5ieme jour à compter de l'ensemencement des cellules épithéliales prolifératives. La présente invention concerne également un équivalent in vitro de follicule pileux susceptible d'être obtenu selon le procédé précité. In a particular embodiment, 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. The present invention also relates to an in vitro equivalent of hair follicle obtainable by the aforementioned method.
Plus particulièrement, l'équivalent in vitro de follicule pileux est caractérisé en ce qu'il est constitué d'un équivalent de papille dermique présentant une activité phosphatase alcaline positive et de kératinocytes positifs pour les marqueurs K85 et K35 et éventuellement Ki67. L'activité phosphatase alcaline est mesurée selon la méthode précitée au paragraphe « Procédé de préparation d'un équivalent de papille dermique ». More particularly, 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".
Les marquages des kératines spécifiques du cheveu K85, K35 et de la protéine Ki67 marqueur de la prolifération cellulaire sont réalisés selon la méthode d'immunoflurorescence.  The markings of keratin specific to hair K85, K35 and protein Ki67 marker of cell proliferation are carried out according to the immunofluorescence method.
L'équivalent de follicule pileux présente en particulier une structure tubulaire pleine de diamètre compris entre ΙΟΟμιη et 250μιη, et de longueur d'au moins 500μιη, de préférence de longueur allant de 500μιη à 2500 μιη, encore mieux de 1500 μιη à 2500 μιη. Etape préliminaire d'amplification The hair follicle equivalent has in particular a tubular structure with a diameter of between ΙΟΟμιη and 250μιη, and a length of at least 500μιη, preferably of length ranging from 500μιη to 2500μιη, more preferably from 1500μιη to 2500μιη. Preliminary stage of amplification
Le procédé de préparation in vitro d'un équivalent de follicule pileux selon l'invention peut comprendre préalablement à l'étape de culture de cellules épithéliales prolifératives en présence d'au moins un équivalent de papille dermique selon l'invention, une étape préliminaire d'amplification de cellules épithéliales prolifératives en présence d'une quantité efficace d'un inhibiteur de ROCK.  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.
Les cellules épithéliales prolifératives telles que les cellules de la matrice et les cellules germinales sont extraites par microdissection du follicule pileux et sont amplifiées selon la technique de Rheinwald et Green (Cell, vol 6 , 331-344,1975) par culture sur un support nourricier constitué de fîbroblastes dans un milieu adapté connu de l'homme du métier, en présence de facteurs de croissance, notamment d'acides aminés, sérum, toxine cholérique, insuline, tri-iodo-thyronine et solution tampon de pH. En particulier, un tel milieu de culture pourra notamment contenir au moins un facteur de croissance mitogénique pour les kératinocytes (par exemple epidermal growth factor (EGF) et/ou keratinocyte growth factor (KGF), en particulier du KGF), de l'insuline, de l'hydrocortisone et facultativement un antibiotique (ex : gentamycine, amphotericine B) auquel a été ajouté un inhibiteur de Rock.. Les inhibiteurs de ROCK peuvent être choisis parmi : Y27632, Ripasudil, Fasudil, Thiazovivin et leurs mélanges. 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. In particular, 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.
Avantageusement, ledit milieu pourra comprendre en outre du sérum ou un extrait pituitaire, par exemple d'origine bovine, de l'épinephrine, de la transferrine et/ou des acides aminés non essentiels.  Advantageously, said medium may further comprise serum or a pituitary extract, for example of bovine origin, epinephrine, transferrin and / or nonessential amino acids.
Les fîbroblastes utilisés pour cette culture seront plus préférentiellement des fîbroblastes 3T3. Les fîbroblastes 3T3 sont bien connus de l'homme du métier. C'est une lignée cellulaire de fîbroblastes connue depuis 1962. « 3T3 » signifie « 3-day transfer, inoculum 3xl05cells ». La culture des cellules épithéliales prolifératives est préférentiellement une culture sur des fïbroblastes (préférentiellement des fïbroblastes 3T3) dont la prolifération a été préalablement stoppée, préférentiellement en les ayant préalablement irradiés (par exemple, aux rayons gamma) ou préalablement traités à la mitomycine. La mitomycine (en particulier, la mitomycine C) bloque la prolifération de ces cellules sans pour autant les empêcher de produire des substances nutritives utiles pour la prolifération des kératinocytes. 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.
Selon l'invention la quantité efficace de l'inhibiteur de ROCK, en particulier Y27632, est comprise entre 1 et 100 μΜ et de préférence comprise entre 5 et 25 μΜ et de manière préférée de ΙΟμΜ. According to the invention, the effective amount of the ROCK inhibitor, in particular Y27632, is between 1 and 100 μΜ and preferably between 5 and 25 μΜ and preferably ΙΟμΜ.
Selon l'invention les cellules épithéliales sont cultivées en présence de l'inhibiteur de ROCK, en particulier Y27632, pendant au moins 2 jours et de préférence pendant au moins 3 jours. De manière préférée les cellules sont mises en culture en une densité de cellules comprise entre 1000 et 4000 cellules/cm2 et de préférence en une densité de 3000 cellules/cm2. According to the invention 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. Preferably, 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 .
Utilisations uses
Etant donné que les équivalents in vitro de papille dermique et de follicule pileux présentent respectivement la plupart des caractéristiques d'une papille dermique et d'un follicule pileux in vivo, ils pourront être utilisés comme implants, associés ou non à des substituts cutanés.  Since the in vitro equivalents of dermal papilla and hair follicle respectively have most of the characteristics of a dermal papilla and hair follicle in vivo, they can be used as implants, whether or not associated with cutaneous substitutes.
Les équivalents in vitro de papille dermique et de follicule pileux selon l'invention trouveront donc également des applications pour la préparation d'implants et/ou de substituts cutanés pour traiter un désordre cutané tel qu'une brûlure, un défaut de cicatrisation ou la canitie. 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 .
Un effet thérapeutique est défini comme un retour à la normale de l'état de pilosité, que ce soit totalement ou partiellement. A therapeutic effect is defined as a return to the normal state of hair, whether totally or partially.
Au sens de l'invention, le traitement prophylactique est recommandé si le sujet possède une condition préalable pour la perte de cheveux, comme une prédisposition familiale.  For the purposes of the invention, the prophylactic treatment is recommended if the subject has a prerequisite for hair loss, as a family predisposition.
Les conditions d'une quantité réduite de cheveux peuvent être le résultat de l'alopécie, la calvitie héréditaire, des cicatrices, des brûlures ou de blessures accidentelles. Ainsi, la présente invention a également pour objet un équivalent de follicule pileux selon l'invention pour le traitement prophylactique ou thérapeutique d'un état de pilosité réduite. The conditions of a reduced amount of hair can be the result of alopecia, hereditary baldness, scarring, burns or accidental injury. Thus, 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.
Un autre de ses objets est un équivalent de follicule pileux selon l'invention pour le traitement de l'alopécie. Another of its objects is a hair follicle equivalent according to the invention for the treatment of alopecia.
Par ailleurs, la présente invention concerne également un équivalent de papille dermique pour le traitement prophylactique ou thérapeutique d'un état de pilosité réduite ou l'alopécie. Furthermore, the present invention also relates to a dermal papilla equivalent for the prophylactic or therapeutic treatment of a reduced hair condition or alopecia.
Procédé de criblage de nouveaux actifs Method of screening new assets
Les équivalents de papille dermique et follicule pileux selon l'invention permettent de réaliser notamment des cinétiques de pousse des poils ou cheveux et donc toute étude nécessitant de nombreux cheveux vivants et aussi complets que possibles dans un contexte in vivo tel que l'étude du cycle du cheveu et des facteurs capables d'influencer ce cycle allant jusqu'à l'étude d'actifs favorisant la croissance du cheveu, d'actifs permettant de lutter contre la chute des cheveux ou encore d'actifs ralentissant la pousse des poils. 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.
Ainsi, la présente invention concerne en outre l'utilisation d'un équivalent in vitro de follicule pileux selon l'invention pour l'identification de composés modulateurs de la pousse des poils et/ou des cheveux. Thus, 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.
Par ailleurs, la présente invention concerne également un procédé de criblage d'au moins un composé modulateur de la pousse des poils et/ou des cheveux comprenant une étape (a) de mise en contact dudit composé à tester avec un équivalent in vitro de follicule pileux selon l'invention puis une étape (b) d'analyse de l'effet dudit composé sur au moins un paramètre de l'équivalent in vitro de follicule pileux et une étape (c) de sélection du composé modifiant ledit paramètre. De préférence, pour la mise en œuvre de l'étape (a), le composé modulateur à tester est appliqué de façon topique, par exemple, formulé dans des formulations topiques classiques ou bien introduit dans le milieu de culture. L'étape (b) pourra, en particulier, être réalisée par l'analyse de l'expression, de la production et/ou de l'activité de marqueurs liés à la qualité et/ou à l'homéostasie du follicule pileux comme par exemple des marqueurs épidermiques et/ou dermiques, tels que des protéines de structure. Comme exemple de protéines de structure peuvent être citées les kératines du cheveu. Furthermore, 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. Preferably, for the implementation of step (a), 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. As an example of structural proteins can be mentioned keratin hair.
Pour cela, on analysera à l'étape (b) du procédé de criblage l'effet du produit sur la pousse de la tige pilaire. For this purpose, the effect of the product on the growth of the hair shaft will be analyzed in step (b) of the screening method.
L'étape (b) d'analyse de l'effet du produit sera préférentiellement une comparaison d'au moins un paramètre mesuré sur l'équivalent de follicule pileux mis en contact avec le produit à tester à celui ou ceux mesuré(s) sur un équivalent de follicule pileux témoin cultivé dans les mêmes conditions mais qui n'a pas reçu le produit à tester. 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.
L'étape (c) de sélection du produit modifiant le paramètre de l'équivalent de follicule pileux se fera en fonction d'un critère déterminé à l'avance. The step (c) of selecting the product modifying the parameter of the hair follicle equivalent will be based on a criterion determined in advance.
La modification de ce paramètre pourra être une stimulation, une diminution ou une inhibition totale ou partielle de l'expression, de la production et/ou de l'activité desdits marqueurs et/ou de la pousse de la tige pilaire. 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.
Le critère de sélection dudit produit sera par exemple que ce produit à un effet stimulateur ou inhibiteur du paramètre mesuré. The criterion for selecting said product will be, for example, that this product has a stimulating or inhibiting effect on the parameter measured.
L'équivalent de follicule pileux selon l'invention peut aussi être utilisé dans des procédés automatisés de criblages de composés cosmétiques, pharmaceutiques ou dermatologiques pour identifier de nouveaux actifs. 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.
En outre, l'invention se rapporte également à un procédé de criblage d'au moins un composé modulateur de la pousse des poils et/ou des cheveux comprenant une étape (a) de mise en contact dudit composé à tester avec un équivalent in vitro de papille dermique selon l'invention puis une étape (b) d'analyse de l'effet dudit composé sur au moins un paramètre de l'équivalent in vitro de papille dermique et une étape (c) de sélection du composé modifiant ledit paramètre. In addition, 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.
Figures Figure 1 : Localisation des cellules de la matrice et des fïbroblastes utilisés dans le cadre de la présente invention. figures Figure 1: Location of the cells of the matrix and fibroblasts used in the context of the present invention.
Figure 2 : Localisation des cellules germinales.  Figure 2: Location of germ cells.
Figure 3 : Amplification des fïbroblastes issus de papille dermique dans le milieu de culture nutritif A.  Figure 3: Amplification of fibroblasts derived from dermal papilla in the nutrient culture medium A.
Figure 4 : Obtention d'équivalent de papille dermique après culture des fïbroblastes amplifiés dans le milieu de culture nutritif B exempt de sérum.  Figure 4: Obtaining dermal papilla equivalent after culturing amplified fibroblasts in serum-free nutrient culture medium B.
Figure 5 : T+l jour après ensemencement des cellules de la matrice sur les équivalents de papilles dermiques.  Figure 5: T + 1 day after seeding of the cells of the matrix on the dermal papillae equivalents.
Figure 6 : Formation d'une structure tubulaire à l'origine de la formation d'un équivalent de follicule pileux (T+4 jours à compter de l'ensemencement des cellules de la matrice sur les équivalents de papilles dermiques). 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 : Croissance de l'équivalent de follicule pileux (T+10 jours à compter de l'ensemencement des cellules de la matrice sur les équivalents de papilles dermiques).  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 : Activité phosphatase alcaline fortement positive d'équivalent de papille dermique selon l'invention. Figure 8: Highly positive alkaline phosphatase activity of dermal papilla equivalent according to the invention.
Figure 9 : Follicule pileux présentant un marquage positif pour les marqueurs K35, K85 et Ki67. Figure 10 : Comparatif WO2017/055358 : T + 10 jours à compter de l'ensemencement des cellules de la matrice.  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.
Figure 11 : Comparatif hors invention : papille dermique obtenue selon le procédé décrit à l'exemple 2 de WO 2009/118283 (grossissement xlO, J6) FIG. 11: Comparative Excludes Invention: Dermal papilla obtained according to the method described in Example 2 of WO 2009/118283 (magnification x10, J6)
Figure 12 : Equivalent de papille dermique selon l'invention obtenu selon l'exemple 1 sur support de culture 2D (grossissement xlO, J6).  12: Dermal papilla equivalent according to the invention obtained according to Example 1 on a 2D culture support (magnification x10, J6).
Figure 13 : Comparatif hors invention : follicule pileux sous forme de kyste obtenu selon le procédé décrit à l'exemple 3 de WO 2009/118283 (J3).  Figure 13: Comparative except invention: hair follicle cyst obtained according to the method described in Example 3 of WO 2009/118283 (J3).
Figure 14 : Comparatif hors invention : culture de fïbroblastes issus de papille dermique sur un support de culture 2D pour culture cellulaire (i.e. support permettant l'adhésion des cellules). 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).
Figure 15 : Equivalent de papille dermique selon l'invention obtenu selon l'exemple 1 sur support de culture 3D microplaque fond rond (J2). FIG. 15: Dermal papilla equivalent according to the invention obtained according to Example 1 on 3D microplate round bottom culture medium (J2).
Figure 16 : Comparatif hors invention : follicule pileux obtenu en culture 3D gel de collagène. Figure 16: Comparative out of invention: hair follicle obtained in 3D collagen gel culture.
Dans la description et dans les exemples suivants, sauf indication contraire, les plages de valeurs libellées sous la forme « entre ... et ... » incluent les bornes inférieure et supérieure précisées. Au sens de la présente invention, « au moins un » doit se comprendre, sauf indication contraire, au sens de « un ou plusieurs ». In the description and in the following examples, unless otherwise indicated, ranges of values labeled "between ... and ..." include the specified lower and upper bounds. For the purposes of the present invention, "at least one" must be understood, unless otherwise indicated, in the sense of "one or more".
Les exemples figurant ci-après sont présentés à titre illustratif et non limitatif de l'invention. Exemple 1 - Préparation d'un équivalent de papille dermique selon l'invention  The examples given below are presented for illustrative and non-limiting purposes of the invention. Example 1 - Preparation of a dermal papilla equivalent according to the invention
Protocole expérimental i. Microdissection des fibroblastes de la papille dermique Les fibroblastes issus de la papille dermique ont été prélevés selon le procédé qui suit : des follicules pileux en phase anagène, disséqués à partir d'un lifting, sont placés dans une boite de Pétri contenant un milieu de culture minimum additionné de 2% d'antibiotique et des acides aminés non essentiels. (Figure 1). ii. Conditions de culture Experimental protocol i. Microdissection of fibroblasts of the dermal papilla The fibroblasts resulting from the dermal papilla were removed according to the following procedure: hair follicles in the anagen phase, dissected from a facelift, are placed in a petri dish containing a culture medium minimum supplemented with 2% antibiotic and non-essential amino acids. (Figure 1). ii. Culture conditions
• milieu de culture nutritif A pour amplification des fibroblastes : Nutrient culture medium A for amplification of fibroblasts:
Le milieu de culture nutritif A pour amplification des fibroblastes a la composition suivante :  The nutrient culture medium A for amplification of fibroblasts has the following composition:
Détail des milieux commerciaux utilisés pour la préparation du milieu de culture A Detail of the commercial environments used for the preparation of the culture medium A
Milieu DMEM Glutamax (Gibco n° 31966047) Composés Concentration (mg/L)DMEM Medium Glutamax (Gibco No. 31966047) Compounds Concentration (mg / L)
Glycine 30.0 Glycine 30.0
I .-Alanyl-l -Glutamine 862.0  I.-Alanyl-1-Glutamine 862.0
L-Arginine chlorhydrate 84.0  L-Arginine hydrochloride 84.0
L-Cystine 2HC1 63.0  L-Cystine 2HC1 63.0
L-Histidine chlorhydrate -H20 42.0  L-Histidine hydrochloride -H20 42.0
L-Isoleucine 105.0  L-Isoleucine 105.0
L-Leucine 105.0  L-Leucine 105.0
l.-Lysine chlorhydrate 146.0 l.-Lysine hydrochloride 146.0
L-Methionine 30.0  L-Methionine 30.0
L-Phenylalanine 66.0  L-Phenylalanine 66.0
L- Serine 42.0  L-Serine 42.0
L-Threonine 95.0  L-Threonine 95.0
L-Tryptophane 16.0  L-Tryptophan 16.0
L-Tyrosine 72.0  L-Tyrosine 72.0
L-Valine 94.0  L-Valine 94.0
Chlorure de Choline 4.0  Choline Chloride 4.0
D-Calcium pantothenate 4.0  D-Calcium Pantothenate 4.0
Acide Folique 4.0  Folic Acid 4.0
Niacinamide 4.0  Niacinamide 4.0
Chlorhydrate de pyridoxine 4.0 Composés Concentration (mg/L) Pyridoxine hydrochloride 4.0 Compounds Concentration (mg / L)
Riboflavine 0.4 Riboflavin 0.4
Chlorhydrate de Thiamine 4.0 i-inositol 7.2 Thiamine Hydrochloride 4.0 i-inositol 7.2
Chlorure de Calcium (CaC12-2H20) 264.0 Calcium Chloride (CaC12-2H20) 264.0
Nitrate de Fer (Fe(N03)3"9H20) 0.1 Iron Nitrate (Fe (N0 3) 3 "9H 2 O) 0.1
Magnésium Sulfate (MgS04-7H20) 200.0 Magnesium Sulfate (MgS04-7H20) 200.0
Chlorure de potassium (KC1) 400.0 Potassium Chloride (KC1) 400.0
Sodium. Bicarbonate (NaHC03) 3700.0 Sodium. Bicarbonate (NaHC03) 3700.0
Chlorure de Sodium (NaCl) 6400.0 Sodium Chloride (NaCl) 6400.0
Dihydrogénophosphate de sodium (NaH2P04-2H20) 141.0 Sodium Dihydrogenphosphate (NaH2PO4-2H2O) 141.0
D-Glucose (Dextrose) 4500.0 D-Glucose (Dextrose) 4500.0
Phénol Red 15.0 Phenol Red 15.0
Acides aminés non essentiels Gibco n° 1 1 140-035 Non-essential amino acids Gibco No. 1 1 140-035
Composés Concentration (mg/L)  Compounds Concentration (mg / L)
Glycine 750.0 Glycine 750.0
L-Alanine 890.0 L-Alanine 890.0
L-Asparagine 1320.0 L-Asparagine 1320.0
Acide L-Aspartique 1330.0 L-Aspartic Acid 1330.0
Acide L-Glutamique 1470.0 Composés Concentration (mg/L) L-Glutamic acid 1470.0 Compounds Concentration (mg / L)
I. -Praline 1150.0  I. -Praline 1150.0
L- Serine 1050.0 Serine 1050.0
Antibiotiques/Antimycotiques Gibco n° 15240-062 Gibco Antibiotics / Antimycotics # 15240-062
• milieu de culture nutritif B pour la préparation des équivalents de papille dermique Le milieu de culture nutritif B pour la préparation des équivalents de papille dermique a composition suivante : Nutrient culture medium B for the preparation of the dermal papilla equivalents The nutrient culture medium B for the preparation of the dermal papilla equivalents has the following composition:
Détail des milieux commerciaux utilisés pour la préparation du milieu de culture B Detail of the commercial environments used for the preparation of culture medium B
Milieu de Williams E (sans glutamine) (Gibco n° A 12176-01) Williams E medium (without glutamine) (Gibco No. A 12176-01)
Composés Concentration (mg/L)  Compounds Concentration (mg / L)
Glycine 50.0 Composés Concentration (mg/L) l.-Alanine 90.0 Glycine 50.0 Compounds Concentration (mg / L) l.-Alanine 90.0
L-Arginine 50.0  L-Arginine 50.0
L-Asparagine-H20 20.0  L-Asparagine-H20 20.0
Acide L-Aspartique 30.0  L-Aspartic Acid 30.0
L-Cysteine 40.0  L-Cysteine 40.0
L-Cystîne 2HCI. 26.07  L-Cystine 2HCI. 26.07
Acide L-Glutamique 50.0  L-Glutamic acid 50.0
L-Histidine 15.0  L-Histidine 15.0
L-Isoleucine 50.0  L-Isoleucine 50.0
L-I.eucine 75.0  L-Ieucine 75.0
1. -Lysine chlorhydrate 87.46  1. -Lysine hydrochloride 87.46
L-Methionine 15.0  L-Methionine 15.0
L-Phenylalanine 25.0  L-Phenylalanine 25.0
L-Proline 30.0  L-Proline 30.0
L-Serine 10.0  L-Serine 10.0
L-Threonine 40.0  L-Threonine 40.0
L-Tryptophane 10.0  L-Tryptophan 10.0
Sel disodique dihydraté de L-Tyrosine 50.65 Composés Concentration (mg/L) l.-Valine 50.0 Disodium salt dihydrate of L-Tyrosine 50.65 Compounds Concentration (mg / L) l.-Valine 50.0
Acide Ascorbique 2.0  Ascorbic acid 2.0
Biotinc 0.5  Biotinc 0.5
Chlorare de Choline 1.5  Choline Chloride 1.5
D-Calcium. pantothenate 1.0  D-Calcium. pantothenate 1.0
Ergocalciferol 0.1  Ergocalciferol 0.1
Acide Folique 1.0  Folic acid 1.0
Menadione sodium, bisulfate 0.01  Menadione sodium, bisulfate 0.01
Niacmarnide 1.0  Niacmarnide 1.0
Chlorhydrate de pyridoxal 1.0  Pyridoxal Hydrochloride 1.0
Riboflavine 0.1  Riboflavin 0.1
Chlorhydrate de thiamine 1.0  Thiamine hydrochloride 1.0
Vitamine A (acétate) 0.1  Vitamin A (acetate) 0.1
Vitamine B12 0.2  Vitamin B12 0.2
alpha Tocopherol phos. Na sel 0.01 alpha Tocopherol phos. Na salt 0.01
i-Inositol 2.0 i-Inositol 2.0
Chlorure de calcium (CaCl2) (anhyd.) 200.0  Calcium Chloride (CaCl2) (anhyd.) 200.0
Sulfate de cuivre (CuS04-5H20) 1.0E-4 Composés Concentration (mg/L) Copper sulphate (CuS04-5H20) 1.0E-4 Compounds Concentration (mg / L)
Sulfate de fer (FeS04-7H20) l .OH-4 Iron sulphate (FeSO4-7H2O) l .OH-4
Sulfate de magnésium (MgSQ4) (anhyd. ) 97.67 Magnesium sulphate (MgSQ4) (anhyd) 97.67
Sulfate de manganèse ( :nSO4»H20) 1.0E-4 Manganese sulphate (: nSO4 » H20) 1.0E-4
Chlorure de Potassium (KC1) 400.0 Potassium Chloride (KC1) 400.0
Sodium Bicarbonate (NaHCOS) 2200.0 Sodium Bicarbonate (NaHCOS) 2200.0
Chlorure de sodium. (NaCl) 6800.0 Sodium chloride. (NaCl) 6800.0
Dihydrogénophosphate de sodium (NaH2P04) anhydre 140.0 Sodium Dihydrogen Phosphate (NaH2P04) Anhydrous 140.0
Sulfate de Zinc(ZnS04-7H20) 2.01.-4 Zinc Sulphate (ZnS04-7H20) 2.01.-4
D-G lucose (Dextrose) 2000.0 D-G lucose (Dextrose) 2000.0
Glutathione (reduced) 0.05 Glutathione (reduced) 0.05
Methyl linoleate 0.03 Methyl linoleate 0.03
Sodium. Pyruvate 25.0 Sodium. Pyruvate 25.0
Antibiotiques/ Antimycotiques Gibco n° 15240-062 (cf. ci-dessus). Culture- Amplification des fibroblastes de la papille dermique Gibco Antibiotics / Antimycotics # 15240-062 (see above). Culture- Amplification of fibroblasts of the dermal papilla
La papille dermique située dans la région bulbaire du follicule, est repérée sous le microscope. A l'aide d'un scalpel et d'aiguilles, la papille dermique est microdisséquée puis placée dans une boite de culture contenant le milieu de culture nutritif A tel que décrit ci-dessus. (Figure 3). Culture-Préparation de l'équivalent de papille dermique Après amplification des fïbroblastes en culture monocouche, les fïbroblastes sont trypsinés puis déposés dans une boite de Pétri non traitée pour la culture cellulaire (boite de pétri bactériologique Falcon®, Corning, ref : 351007) à forte densité (par exemple 23800 cellules par cm2), dans le milieu de culture nutritif B exempt de sérum tel que décrit ci-dessus. 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.
Les fïbroblastes migrent dans la boite de Pétri et se regroupent pour former des clusters, puis des agrégats cellulaires, pour enfin se détacher du support afin de former des sphéroïdes ou équivalents de papilles dermiques après 5 jours de culture. (Figures 4 et 12). Les papilles dermiques obtenues sont de forme sphérique d'environ 200μιη de diamètre. 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. (Figures 4 and 12). The dermal papillae obtained are spherical in shape of about 200 μm in diameter.
Un marquage de l'activité enzymatique à la phosphatase alcaline est réalisé à l'aide du kit phosphatase alcaline NBT/BCIP (Roche réf : 11 681 451 001) où le BCIP (sel de 5-Bromo-4- chloro-3-indolyl phosphate toluidine), substrat de la phosphatase alcaline, sera d'abord déphosphorylé puis oxydé pour donner un produit coloré bleu. 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.
Une coloration violet foncé intense montre une activité enzymatique à la phosphatase alcaline fortement positive. (Figure 8). On a également réalisé des équivalents de papille dermique selon l'invention obtenus selon le même procédé que celui de l'Exemple 1 en remplaçant : Intense dark purple coloration shows strongly positive alkaline phosphatase enzymatic activity. (Figure 8). Dermal papilla equivalents according to the invention were also obtained, obtained according to the same method as in Example 1, replacing:
le support 2D (boite de pétri bactériologique Falcon®, Corning) par le support 3D microplaque 96 puits à fond rond commercialisé sous le nom de costar® par la société Corning ;  the 2D support (bacterial bacteriological box Falcon®, Corning) by the 3D round-bottom 96 well microplate medium marketed under the name of costar® by Corning;
- la densité d'ensemencement des fïbroblastes de 23800 cellules/cm2 par 9375 cellules/cm2. the fibroblast seeding density of 23800 cells / cm 2 by 9375 cells / cm 2 .
Les papilles dermiques obtenues sont également de forme sphérique d'environ 200μιη de diamètre (voir Figure 15) et présentent une activité enzymatique phosphatase alcaline fortement positive. 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.
Conclusion : Les papilles dermiques selon l'invention ainsi obtenues présentent bien les caractéristiques morphologiques et fonctionnelle d'une papille dermique in vivo, Exemple 2 - Préparation d'un équivalent de follicule pileux selon l'invention Protocole expérimental i. Microdissection des cellules de la matrice Conclusion: 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
Les follicules pileux sont extraits d'un résidu chirurgical de scalp. Ce dernier est d'abord coupé en portions de 5 mm2 puis sectionné à l'aide d'un scalpel entre le derme et Phypoderme. 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.
Les follicules sont extraits à l'aide de pinces de chirurgie ophtalmique et sont ensuite sectionnés juste au-dessus de la papille avec un scalpel. Le bulbe est alors récupéré. A cette étape, le bulbe comprend deux compartiments : Le compartiment dermique (papille dermique et gaine conjonctive) et les cellules de la matrice qui forment une masse cellulaire. (Figure 1). The follicles are extracted using ophthalmic surgery forceps and are then cut just above the disc with a scalpel. The bulb is then recovered. At this stage, 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).
A l'aide d'aiguilles à perfusion, la partie épithéliale est séparée de la partie dermique. ii. Conditions de culture With the help of infusion needles, the epithelial part is separated from the dermal part. ii. Culture conditions
Les conditions de culture ont trois composantes principales : The growing conditions have three main components:
• Le milieu de base : • The basic medium:
Sauf indication contraire, l'ensemble des milieux et tampons utilisés dans les exemples sont décrits dans Bell et col. 1979, (P.N.A.S. USA, 76, 1274-1278), Asselineau et Prunieras, 1984, (British J. of Demi., 111, 219-222) ou Asselineau et col, 1987, (Models in dermato., vol.III, Ed. Lowe & Maibach, 1-7).  Unless otherwise indicated, all media and buffers used in the examples are described in Bell et al. 1979, (PNAS USA, 76, 1274-1278), Asselineau and Prunieras, 1984, (British J. of Demi., 111, 219-222) or Asselineau et al, 1987, (Models in dermato., Vol. Ed. Lowe & Maibach, 1-7).
Le milieu DMEM + 10% FCS + 7F (appelé milieu G7F) a la composition suivante : DMEM + 10% FCS + 7F medium (called G7F medium) has the following composition:
Concentrations finales Final concentrations
DMEM 500 ml  DMEM 500 ml
Sérum de veau foetal (FCS) 10%  Fetal calf serum (FCS) 10%
L-Glutamine 2 mM  L-Glutamine 2 mM
Sodium pyruvate 1 mM  Sodium pyruvate 1 mM
Penicicilline - Steptomycine Pénicilline 20 U/ml  Penicicillin - Steptomycin Penicillin 20 U / ml
Streptomycine 20 μg/ml Fungizone Pénicilline 10 U/ml Streptomycin 20 μg / ml Fungizone Penicillin 10 U / ml
Streptomycine 10 μg/ml  Streptomycin 10 μg / ml
Amphotericine-B 25 ng/ml  Amphotericin-B 25 ng / ml
Epidermal growth factor (EGF) 10 ng/ml  Epidermal growth factor (EGF) 10 ng / ml
Choiera toxine 10"10 M Choiera toxin 10 " 10M
Hydro cortisone 0.4 μg/ml  Hydro cortisone 0.4 μg / ml
Adénine hydrochloride 1.8 x 10"4 M Adenine hydrochloride 1.8 x 10 "4 M
Triiodothyonine (T3) 2 x 10"9 M Triiodothyonine (T3) 2 x 10 "9 M
Transferrine humaine 5 μg/ml  Human transferrin 5 μg / ml
Insuline bovine 5 μg/ml  Bovine insulin 5 μg / ml
• Les compléments de culture : 10 μΜ de Y27632. • The cultivation complements: 10 μΜ of Y27632.
• La surface d'adhésion : les cellules de la matrice adhèrent et prolifèrent dans le milieu de base Green en présence d'une couche nourricière de fïbroblastes murins 3T3 arrêtés dans le cycle cellulaire par un traitement mitomycine.  • 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.
Culture- Amplification des cellules de la matrice Après microdissection, les amas de cellules de la matrice sont déposés dans des boîtes de Pétri de 60mm de diamètre préalablement ensemencées d'un feeder layer, de préférence avec 40 000 3T3irradié/cm2 et recouverte du milieu de culture complet, par exemple le milieu G7F +10μΜ Y27632 ; on obtient une culture à 70% de confluence de cellules de la matrice. Culture-Préparation de l'équivalent de follicule pileux Culture- Amplification of the cells of the matrix After microdissection, the clusters of cells of the matrix are deposited in Petri dishes 60 mm in diameter previously seeded with a feeder layer, preferably with 40 000 3T3irradié / cm 2 and covered with the medium. complete culture, for example G7F + 10μΜ medium Y27632; a culture at 70% confluence of cells of the matrix is obtained. Culture-Preparation of the equivalent of hair follicle
Afin de générer les équivalents in vitro de follicule pileux, les cellules sont récupérées au stade de sous confluence par un traitement enzymatique. Les cellules sont ensuite ensemencées dans des boites de Pétri bactériologique non traitée pour la culture cellulaire (boite de pétri bactériologique Falcon®, Corning, ref : 351007), contenant préalablement les équivalents de papilles dermiques, à une densité de 6000 cellules / cm2, dans le milieu de culture nutritif B exempt de sérum tel que décrit à l'Exemple 1. In order to generate the in vitro equivalents of hair follicle, 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.
Les cellules de la matrice adhérent uniquement au niveau des sphéroïdes de papilles dermiques et prolifèrent. (Figures 5 et 6). Après 6 jours de co-culture, on voit apparaître des équivalents de follicule pileux, présentant en particulier les caractéristiques morphologiques suivantes : structure tabulaire pleine de diamètre d'environ 2500 micromètres. (Figures 6 et 7). The cells of the matrix adhere only to the level of the dermal papillae spheroids and proliferate. (Figures 5 and 6). After 6 days of co-culture, equivalents of hair follicle appear, having in particular the following morphological characteristics: full tubular structure of diameter of about 2500 micrometers. (Figures 6 and 7).
Le marquage des kératines spécifiques du cheveu K85 et K35, ainsi qu'un marqueur de prolifération cellulaire, comme le Ki67, est réalisé par immunomarquage en fluorescence. (Figure 9).  The labeling of hair-specific keratins K85 and K35, as well as a cell proliferation marker, such as Ki67, is carried out by fluorescence immunolabeling. (Figure 9).
Conclusion : Les follicules pileux selon l'invention ainsi obtenus présentent bien les caractéristiques morphologiques et fonctionnelle d'un follicule pileux in vivo. Conclusion: The hair follicles according to the invention thus obtained have the morphological and functional characteristics of a hair follicle in vivo.
Exemple 3 : Comparatif hors invention : papille dermique obtenue selon le procédé décrit à l'exemple 2 de WO 2009/118283 EXAMPLE 3 Comparative Excluding Invention: Dermal Papilla Obtained by the Method Described in Example 2 of WO 2009/118283
IX Matériel et Méthode : IX Material and Method:
La préparation de l'équivalent de papille dermique a été réalisée selon le protocole de préparation décrit à l'exemple 2 de WO 2009/118283. The preparation of the dermal papilla equivalent was carried out according to the preparation protocol described in Example 2 of WO 2009/118283.
Préparation du milieu DMEM (+): Preparation of the DMEM (+) medium:
- 500 ml de DMEM Glutamax (Invitrogen n°31966)  500 ml of DMEM Glutamax (Invitrogen No. 31966)
- 5 ml Acide aminé non essentiels (AANE) (Gibco n° 11140-035)  - 5 ml Non-essential amino acid (AANE) (Gibco No. 11140-035)
- 50 μΐ d'Acide ascorbique à lOOmM (Sigma n°A8960), soit 2,9mg/L final  50 μl of Ascorbic acid at 100 mM (Sigma No. A8960), ie 2.9 mg / L final
5ml de Insuline-Transferrin-Sodium Selenite (ITS) (Fisher Scientific n° 10524233) 5ml of Insulin-Transferrin-Sodium Selenite (ITS) (Fisher Scientific No. 10524233)
- 400 mg/L BSA - 400 mg / L BSA
Support de culture : 3D Plaque 6 puits ULA (ultra low attachment) fond plat. Culture support: 3D Plate 6 wells ULA (ultra low attachment) flat bottom.
Densité cellulaire : 6660 cellules/cm2. Cell density: 6660 cells / cm 2 .
Cellules : fîbroblastes issus de papille dermique (passage P6). 2} Résultats : (voir Figure 11) Cells: fibroblasts from the dermal papilla (passage P6). 2} Results: (see Figure 11)
Conclusion : on observe des agrégats cellulaires de différentes tailles, très hétérogènes, à bords irréguliers. Exemple 4 : Comparatif hors invention : papille dermique obtenue selon le procédé décrit à l'exemple 3 de WO 2009/118283 Conclusion: we observe cell aggregates of different sizes, very heterogeneous, with irregular edges. EXAMPLE 4 Comparative Excluding Invention: Dermal Papilla Obtained by the Method Described in Example 3 of WO 2009/118283
IX Matériel et Méthode : IX Material and Method:
La préparation de l'équivalent follicule pileux a été réalisée selon le protocole de préparation décrit à l'exemple 3 de WO 2009/118283. The preparation of the hair follicle equivalent was performed according to the preparation protocol described in Example 3 of WO 2009/118283.
Milieux de culture : Culture media:
- DMEM (+) pour les équivalent de papilles (DMEM+2%BSA+ITS+Vit C)  - DMEM (+) for the taste buds (DMEM + 2% BSA + ITS + Vit C)
- Ensuite KSFM pour la culture mixte DP/MHN/ORS  - Then KSFM for mixed culture DP / MHN / ORS
Support de culture : Plaque 6 puits ULA (ultra low attachment) fond plat. Densité cellulaire : Culture medium: 6-well ULA plate (ultra low attachment) flat bottom. Cell density:
Pour la préparation de la papille dermique : fïbroblastes issus de papille dermique : 500 000 DP/F75 soit 6660 DP/cm2 ; For the preparation of the dermal papilla: fibroblasts from the dermal papilla: 500 000 DP / F75 ie 6660 DP / cm 2 ;
- Pour la préparation du follicule pileux : 250 000 ORS/6 puits soit 26300 ORS/cm2 + 25 000 MHN/6 puits soit 2630 MHN/cm2. - For the preparation of the hair follicle: 250 000 ORS / 6 well is 26300 ORS / cm 2 + 25 000 MHN / 6 wells or 2630 MHN / cm 2 .
Cellules : Cells:
Fïbroblastes issus de papille dermique (passage P6)  Fibroblasts from the dermal papilla (passage P6)
Mélanocytes M597 (passage P4)  Melanocytes M597 (passage P4)
Kératinocytes de l'ORS IB (passage P3)  ORS IB Keratinocytes (passage P3)
2} Résultats (voir Figure 13) 2} Results (see Figure 13)
Conclusion : on observe des agrégats cellulaires (kystes) sans évolution vers un follicule pileux. Conclusion: Cell aggregates (cysts) are observed without evolution towards a hair follicle.
Exemple 5 : Comparatif hors invention : culture de fïbroblastes issus de papille dermique sur un support de culture 2D pour culture cellulaire (i.e. support permettant l'adhésion des cellules). 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).
H Matériel et Méthode La préparation de l'équivalent de papille dermique a été réalisée selon le protocole de préparation décrit à l'exemple 1, en remplaçant le support de culture boite de pétri bactériologique Falcon®, Corning, ref : 351007 par un support de culture boite de pétri pour culture cellulaire (i.e. permettant l'adhésion cellulaire). H Material and Method The preparation of the dermal papilla equivalent was carried out according to the preparation protocol described in Example 1, replacing the Falcon® bacterial bacterial culture dish support, Corning, ref: 351007 with a petri dish culture support. for cell culture (ie allowing cell adhesion).
2} Résultats : (voir Figure 2} Results: (see Figure
Conclusion : On n'observe pas de formation de papille dermique en utilisant un support de culture permettant l'adhésion des cellules. Conclusion: No dermal papilla formation is observed using a culture support allowing cell adhesion.
Exemple 6 : Comparatif hors invention : papille dermique obtenue dans un milieu de culture contenant du sérum tel que décrit dans Higgins et al. {« Modelling the haïr follicle dermal papilla using spheroid cell cultures ») 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")
1) Matériel et Méthode 1) Material and Method
Comparatif hors invention : Comparative outside the invention:
- Support de culture 3D : Plaque 96 puits ULA fond U  - 3D culture support: Plate 96 wells ULA bottom U
- Milieu de culture : Milieu DMEM + 10%SVF  - Culture medium: DMEM medium + 10% FCS
Densité cellulaire : 9375 cellules/cm2 Cell Density: 9375 cells / cm 2
Equivalent de papille dermique selon l'invention : Dermal papilla equivalent according to the invention:
- Support de culture 3D : Plaque 96 puits ULA fond U  - 3D culture support: Plate 96 wells ULA bottom U
- Milieu de culture : Milieu Williams (sans sérum)  - Culture medium: Williams medium (serum-free)
- Densité cellulaire : 9375 cellules/cm2 Cell Density: 9375 cells / cm 2
Afin de mesurer l'expression en ARN de la phosphatase alcaline, du Versican, et SFRP2 secreted frizzled related protein 2, qui sont des marqueurs de l'activité enzymatique de la papille dermique on a utilisé les sondes ALPL-HsO 1029144_ml , VCAN-HsOO 171642_ml , et SFRP2- Hs00293258 ml . Variations d'expression In order to measure the RNA expression of alkaline phosphatase, Versican, and SFRP2 secreted frizzled related protein 2, which are markers of the enzymatic activity of the dermal papilla, probes ALPL-HsO 1029144_ml, VCAN-HsOO were used. 171642_ml, and SFRP2-Hs00293258 ml. Variations of expression
Sondes DP P5 3D DP P5 3D  DP P5 3D DP P5 3D Probes
DMEM+10%SVF WILLIAMS E  DMEM + 10% SVF WILLIAMS E
(hors invention) (selon  (except invention) (according to
l'invention)  the invention)
ALPL-Hs01029144_ml 1,00 1.75  ALPL-Hs01029144_ml 1,00 1.75
VC AN-Hs00171642_m 1 1.00 3.61  VC AN-Hs00171642_m 1 1.00 3.61
SFRP2-Hs00293258_ml 1.00 2.18  SFRP2-Hs00293258_ml 1.00 2.18
Conclusion : Conclusion:
Pour l'équivalent de papille dermique selon l'invention, on observe une surexpression des marqueurs ALPL (phosphatase alcaline), VCAN (versican) et SFRP2 (secreted frizzled related protein 2), connus comme marqueur d'inductivité de l'activité enzymatique de la papille dermique.  For the dermal papilla equivalent according to the invention, there is an overexpression of ALPL (alkaline phosphatase), VCAN (versican) and SFRP2 (secreted frizzled related protein 2) markers, known as inductivity markers for the enzymatic activity of the dermal papilla.
Pour le comparatif hors invention, on observe une sousexpression des marqueurs ALPL (phosphatase alcaline), VCAN (versican) et SFRP2 (secreted frizzled related protein 2), connus comme marqueur d'inductivité de l'activité enzymatique de la papille dermique.  For the comparative outside the invention, there is a sub-expression of the ALPL (alkaline phosphatase), VCAN (versican) and SFRP2 (secreted frizzled related protein 2) markers, known as inductivity markers for the enzymatic activity of the dermal papilla.
Exemple 7 : Comparatif hors invention : follicule pileux obtenu en culture 3D gel de collagène EXAMPLE 7 Comparison Compared to Invention: Hair Follicle Obtained in 3D Collagen Gel Culture
IX Matériel et Méthode : On a réalisé une lere étape de fabrication d'un sphéroïde comprenant des fïbroblastes issus de papille dermique et de cellules épithéliales prolifératives (cellules de la matrice), en milieu DMEM+10% sérum, dans une plaque 96 puits ULA fond U. 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.
On a ensuite réalisé une 2e étape qui consiste en l'intégration des sphéroïdes dans un gel de collagène (lattice), l'observation est faite à J14. Was then conducted a 2nd step in the integration of the spheroids in a collagen gel (lattice), observation is made on D14.
2X Résultats : (voir Figure 16) 2X Results: (see Figure 16)
Conclusion : On n'observe pas de formation de follicule pileux dans le gel de collagène, on observe un kyste sans formation de structure apicale. Conclusion: No hair follicle formation is observed in the collagen gel, a cyst without formation of apical structure is observed.

Claims

REVENDICATIONS
1. Procédé de préparation in vitro d'un équivalent de papille dermique comprenant au moins une étape de mise en culture de fïbroblastes issus de la papille dermique et/ou de la gaine conjonctive sur un support comprenant un milieu de culture nutritif B exempt de sérum pendant une période de temps suffisante pour permettre auxdits fïbroblastes de se détacher dudit support et de se regrouper pour former au moins un sphéroïde ; la surface dudit support utilisé ne permettant pas l'adhésion des cellules ; ledit support de culture est choisi parmi les supports de culture 2D ou 3D microplaque fond rond. 1. Process for the in vitro preparation of a dermal papilla equivalent comprising at least one step of culturing fibroblasts derived 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 to aggregate 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.
2. Procédé selon la revendication 1, dans lequel lesdits fïbroblastes sont ensemencés sur ledit support de culture 2D en une densité d'au moins 14000 cellules/cm2, de préférence en une densité comprise entre 14000 cellules/cm2 et 47600 cellules/cm2, encore mieux entre 23500 cellules/cm2 et 24500 cellules/cm2. 2. The method according to claim 1, wherein said fibroblasts are seeded on said 2D culture support in a density of at least 14000 cells / cm 2 , preferably in a density of between 14000 cells / cm 2 and 47600 cells / cm. 2 , even better between 23500 cells / cm 2 and 24500 cells / cm 2 .
3. Procédé selon la revendication 1, dans lequel lesdits fïbroblastes sont ensemencés sur ledit support de culture 3D en une densité d'au moins d'au moins 3000 cellules/cm2, de préférence d'au moins 9000 cellules/cm2, encore mieux en une densité comprise entre 9000 cellules/cm2 et 20 000 cellules/cm2. The method of claim 1, wherein said fibroblasts are seeded on said 3D culture support in a density of at least at least 3000 cells / cm 2 , preferably at least 9000 cells / cm 2 , further better in a density of between 9000 cells / cm 2 and 20 000 cells / cm 2 .
4. Procédé selon l'une quelconque des revendications 1 à 3 dans lequel ledit milieu de culture nutritif B comprend de 500 à 1500 mg/L d'acides aminés, de 2 à 18 mg/L de vitamines, de 1500 à 4500 mg/L de glucose, de 8750 à 10000 mg/L de sels inorganiques, de 2 à 20 μg/ml d'insuline, de 2 à 60 ng/ml d'hydrocortisone, et éventuellement de de 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotiques. The method according to any one of claims 1 to 3 wherein 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 μg / ml of insulin, from 2 to 60 ng / ml of hydrocortisone, and possibly from 50 to 200 μg / ml of antibiotics and / or antimycotics.
5. Procédé selon l'une quelconque des revendications 1 à 4 dans lequel lesdits fïbroblastes sont cultivés pendant au moins 3 jours, de préférence pendant au moins 4 jours, encore mieux entre 4 et 21 jours. 5. A method according to any one of claims 1 to 4 wherein said fibroblasts are cultured for at least 3 days, preferably for at least 4 days, more preferably between 4 and 21 days.
6. Procédé selon l'une quelconque des revendications 1 à 5 dans lequel la surface dudit support est neutre et hydrophobe. 6. Method according to any one of claims 1 to 5 wherein the surface of said support is neutral and hydrophobic.
7. Procédé selon l'une quelconque des revendications 1 à 6 comprenant en outre préalablement à l'étape de mise en culture desdits fïbroblastes les étapes préliminaires suivantes : 7. Method according to any one of claims 1 to 6 further comprising prior to the step of culturing said fibroblasts the following preliminary steps:
a. isoler un follicule pileux en phase anagène à partir d'un prélèvement de scalp ; b. récupérer les fïbroblastes de la papille dermique et/ou de la gaine conjonctive à l'aide d'une microdissection de la papille dermique et/ou de la gaine conjonctive ;  at. isolate a hair follicle in the anagen phase from a scalp sample; b. recover fibroblasts from the dermal papilla and / or conjunctiva sheath using a microdissection of the dermal papilla and / or conjunctiva sheath;
c. effectuer une amplification desdits fïbroblastes de la papille dermique et/ou de la gaine conjonctive dans un milieu de culture nutritif A constitué de DMEM Glutamax supplémenté de 20% en volume de sérum fœtal de veau (S VF), de 50 à 90 mg/L d'acides aminés non essentiels, et éventuellement de 50 à 200 μg/ml d'antibiotiques et/ou d'antimycotique.  c. amplifying said fibroblasts of the dermal papilla and / or the conjunctive sheath in a nutrient culture medium A consisting of DMEM Glutamax supplemented with 20% by volume of fetal calf serum (S VF), from 50 to 90 mg / L non-essential amino acids, and possibly from 50 to 200 μg / ml of antibiotics and / or antimycotic.
8. Equivalent in vitro de papille dermique susceptible d'être obtenu par le procédé selon l'une quelconque des revendications 1 à 7. 8. In vitro equivalent of dermal papilla obtainable by the method according to any one of claims 1 to 7.
9. Equivalent in vitro de papille dermique selon la revendication 8 caractérisée en ce qu'il présente une activité phosphatase alcaline positive. 9. In vitro equivalent of dermal papilla according to claim 8 characterized in that it has a positive alkaline phosphatase activity.
10. Utilisation d'un équivalent in vitro de papille dermique selon l'une quelconque des revendications 8 à 9 et de cellules épithéliales prolifératives pour la préparation d'un équivalent in vitro de follicule pileux. Use of an in vitro equivalent of a dermal papilla according to any of claims 8 to 9 and proliferative epithelial cells for the preparation of an in vitro equivalent of hair follicle.
11. Procédé de préparation in vitro d'un équivalent de follicule pileux comprenant au moins une étape de culture de cellules épithéliales prolifératives en présence d'au moins un équivalent de papille dermique tel que défini selon la revendication 8 ou 9 pendant une période de temps suffisante pour permettre une différenciation desdites cellules épithéliales prolifératives en kératinocytes positifs pour les marqueurs K85 et K35. 11. 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 as defined according to claim 8 or 9 for a period of time sufficient to allow differentiation of said proliferative epithelial cells into keratinocytes positive for K85 and K35 markers.
12. Procédé selon la revendication 11, dans lequel les cellules épithéliales prolifératives sont ensemencées en une densité d'au moins 2000 cellules/cm2, de préférence d'au moins 6000 cellules/cm2, et encore mieux en une densité comprise entre 6000 et 10000 cellules/cm2- The method according to claim 11, wherein the proliferative epithelial cells are seeded at a density of at least 2000 cells / cm 2 , preferably at least 6000 cells / cm 2 , and most preferably at a density of between 6000 and 10,000 cells / cm 2 -
13. Procédé selon la revendication 11 ou 12, dans lequel les cellules épithéliales prolifératives sont cultivées pendant au moins 3 jours, de préférence pendant au moins 5 jours, encore mieux entre 5 et 20 jours. The method of claim 11 or 12, wherein the proliferative epithelial cells are cultured for at least 3 days, preferably for at least 5 days, more preferably between 5 and 20 days.
14. Procédé selon l'une quelconque des revendications 11 à 13 comprenant en outre une étape préliminaire d'amplification desdites cellules épithéliales prolifératives en présence d'une quantité efficace d'un inhibiteur de ROCK. The method of any one of claims 11 to 13 further comprising a preliminary step of amplifying said proliferative epithelial cells in the presence of an effective amount of a ROCK inhibitor.
15. Equivalent in vitro de follicule pileux susceptible d'être obtenu par le procédé selon l'une quelconque des revendications 11 à 14. 15. In vitro equivalent of hair follicle obtainable by the method according to any one of claims 11 to 14.
16. Equivalent in vitro de follicule pileux selon la revendication 15 caractérisé en ce qu'il est constitué d'un équivalent de papille dermique présentant une activité phosphatase alcaline positive et de kératinocytes positifs pour les marqueurs K85 et K35. 16. In vitro equivalent hair follicle according to claim 15 characterized in that it consists of a dermal papilla equivalent with positive alkaline phosphatase activity and positive keratinocytes for markers K85 and K35.
17. Equivalent in vitro de follicule pileux selon la revendication 15 ou 16 caractérisé en ce qu'il présente une structure tubulaire pleine de diamètre allant de 100 à 250μιη, et de longueur allant de 500 à 2500μιη. 17. In vitro equivalent hair follicle according to claim 15 or 16 characterized in that it has a solid tubular structure with a diameter ranging from 100 to 250μιη, and length ranging from 500 to 2500μιη.
18. Equivalent in vitro de follicule pileux selon l'une des revendications 15 à 17 pour le traitement prophylactique ou thérapeutique d'un état de pilosité réduite. 18. In vitro equivalent of hair follicle according to one of claims 15 to 17 for the prophylactic or therapeutic treatment of a state of reduced hairiness.
19. Equivalent in vitro de follicule pileux selon l'une des revendications 15 à 17 pour le traitement de l'alopécie. 19. In vitro equivalent of hair follicle according to one of claims 15 to 17 for the treatment of alopecia.
20. Utilisation d'un équivalent in vitro de follicule pileux selon l'une des revendications 15 à 17 pour l'identification de composés modulateurs de la pousse des poils et/ou des cheveux. 20. Use of an in vitro equivalent of hair follicle according to one of claims 15 to 17 for the identification of compounds modulating the growth of hair and / or hair.
21. Procédé de criblage d'au moins un composé modulateur de la pousse des poils et/ou des cheveux comprenant une étape (a) de mise en contact dudit composé à tester avec un équivalent in vitro de follicule pileux selon l'une quelconque des revendications 15 à 17 puis une étape (b) d'analyse de l'effet dudit composé sur au moins un paramètre de l'équivalent in vitro de follicule pileux et une étape (c) de sélection dudit composé modifiant ledit paramètre. 21. A method of 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 hair follicle according to any one of 15 to 17 and 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 said compound modifying said parameter.
EP18803336.9A 2017-10-30 2018-10-30 Method for the in vitro preparation of dermal papilla and hair follicle equivalents Pending EP3704226A1 (en)

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