METHOD FOR THE IN VITRO PREPARATION OF DERMAL PAPILLA AND HAIR FOLLICLE EQUIVALENTS

Abstract

The invention relates to a process for the in vitro preparation of a dermal papilla equivalent from fibroblasts derived from the dermal papilla and/or from the connective tissue sheath; to a process for the in vitro preparation of a hair follicle equivalent by culturing proliferative epithelial cells on said dermal papillae thus obtained; to the in vitro dermal papilla and hair follicle equivalents produced by means of the abovementioned processes, and to the uses thereof for treating alopecia and for evaluating the effect of cosmetic, pharmaceutical or dermatological products.

Claims

1. A 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 from the connective tissue sheath on a support comprising a serum-free nutritive culture medium B for a period of time that is sufficient to allow said fibroblasts to detach from said support and to group together to form at least one spheroid; the surface of said support used not permitting cell adhesion; said culture support is chosen from 2D or 3D round-bottomed microplate culture supports.

2. The process as claimed in claim 1, in which said fibroblasts are seeded on said 2D culture support at a density of at least 14 000 cells/cm.sup.2.

3. The process as claimed in claim 1, in which said fibroblasts are seeded on said 3D culture support at a density of at least 3000 cells/cm.sup.2.

4. The process as claimed in claim 1, in which said nutritive 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 10 000 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 of antimycotics.

5. The process as claimed in claim 1, in which said fibroblasts are cultured for at least 3 days.

6. The process as claimed in claim 1, in which the surface of said support is neutral and hydrophobic.

7. The process as claimed in claim 1, also comprising, prior to the step of culturing said fibroblasts, the following preliminary steps: a. isolating an anagen-phase hair follicle from a scalp sample; b. recovering the fibroblasts of the dermal papilla and/or of the connective tissue sheath by means of microdissection of the dermal papilla and/or of the connective tissue sheath; c. performing an amplification of said dermal papilla fibroblasts and/or of said connective tissue sheath fibroblasts in a nutritive culture medium A consisting of DMEM Glutamax supplemented with 20% by volume of fetal calf serum (FCS), 50 to 90 mg/l of nonessential amino acids, and optionally 50 to 200 g/ml of antibiotics and/or of antimycotics.

8. An in vitro dermal papilla equivalent which may be obtained by means of the process as claimed in claim 1.

9. The in vitro dermal papilla equivalent as claimed in claim 8, which has positive alkaline phosphatase activity.

10. A method preparing an in vitro hair follicle equivalent which comprises using the in vitro dermal papilla equivalent as claimed in claim 8 and of proliferative epithelial cells.

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 in claim 8 for a period of that is time sufficient to allow differentiation of said proliferative epithelial cells into keratinocytes positive for the markers K85 and K35.

12. The process as claimed in claim 11, in which the proliferative epithelial cells are seeded at a density of at least 2000 cells/cm.sup.2.

13. The process as claimed in claim 11, in which the proliferative epithelial cells are cultured for at least 3 days.

14. The process as claimed in claim 11, also comprising a preliminary step of amplifying said proliferative epithelial cells in the presence of an effective amount of a ROCK inhibitor.

15. An in vitro hair follicle equivalent which may be obtained by means of the process as claimed in claim 11.

16. The in vitro hair follicle equivalent as claimed in claim 15, which is constituted of a dermal papilla equivalent having a positive alkaline phosphatase activity and of keratinocytes positive for the markers K85 and K35.

17. The in vitro hair follicle equivalent as claimed in claim 15, wherein it has a solid tubular structure with a diameter ranging from 100 to 250 m, and a length ranging from 500 to 2500 m.

18. The in vitro hair follicle equivalent as claimed in claim 15, for the prophylactic or therapeutic treatment of a state of reduced pilosity.

19. The in vitro hair follicle equivalent as claimed in claim 15, for the treatment of alopecia.

20. A process for identifying compounds which modulate the growth of bodily hair and/or head hair which comprises using the in vitro hair follicle equivalent as claimed in claim 15.

21. A process for screening for at least one compound which modulates the growth of bodily hair and/or head hair, comprising a step (a) of bringing said test compound into contact with an in vitro hair follicle equivalent as claimed in claim 15, then a step (b) of analyzing the effect of said compound on at least one parameter of the in vitro hair follicle equivalent and a step (c) of selecting said compound which modifies said parameter.

Description

FIGURES

[0149] FIG. 1: Localization of the matrix cells and of the fibroblasts used in the context of the present invention.

[0150] FIG. 2: Localization of the germ cells.

[0151] FIG. 3: Amplification of the fibroblasts derived from a dermal papilla in the nutritive culture medium A.

[0152] FIG. 4: Production of a dermal papilla equivalent after culture of the amplified fibroblasts in the serum-free nutritive culture medium B.

[0153] FIG. 5: T+1 day after seeding of the matrix cells on the dermal papilla equivalents.

[0154] FIG. 6: Formation of a tubular structure responsible for the formation of a hair follicle equivalent (T+4 days starting from the seeding of the matrix cells on the dermal papilla equivalents).

[0155] FIG. 7: Growth of the hair follicle equivalent (T+10 days starting from the seeding of the matrix cells on the dermal papilla equivalents).

[0156] FIG. 8: Strongly positive alkaline phosphatase activity of a dermal papilla equivalent according to the invention.

[0157] FIG. 9: Hair follicle showing positive labeling for the markers K35, K85 and Ki67.

[0158] FIG. 10: Comparative WO 2017/055358: T+10 days starting from the seeding of the matrix cells.

[0159] FIG. 11: Comparative outside the invention: dermal papilla obtained according to the process described in example 2 of WO 2009/118283 (magnification 10, D6)

[0160] FIG. 12: Dermal papilla equivalent according to the invention obtained according to example 1 on a 2D culture support (magnification 10, D6).

[0161] FIG. 13: Comparative outside the invention: hair follicle in cyst form obtained according to the process described in example 3 of WO 2009/118283 (D3)

[0162] FIG. 14: Comparative outside the invention: culture of fibroblasts obtained from dermal papilla on a 2D culture support for cell culturing (i.e. support permitting cell adhesion).

[0163] FIG. 15: Dermal papilla equivalent according to the invention obtained according to example 1 on a 3D round-bottomed microplate culture support (D2).

[0164] FIG. 16: Comparative outside the invention: hair follicle obtained in 3D culturing on collagen gel.

[0165] In the description and the examples that follow, unless otherwise indicated, the ranges of values written in the form between . . . and . . . include the stated lower and upper limits.

[0166] For the purposes of the present invention, the term at least one should be understood, unless otherwise indicated, as meaning one or more.

[0167] The examples given below are presented as nonlimiting illustrations of the invention.

EXAMPLE 1PREPARATION OF A DERMAL PAPILLA EQUIVALENT ACCORDING TO THE INVENTION

Experimental Protocol

[0168] i. Microdissection of the Dermal Papilla Fibroblasts

[0169] The fibroblasts derived from the dermal papilla were sampled according to the process which follows: anagen-phase hair follicles, dissected from a facelift, are placed in a Petri dish containing a minimum culture medium supplemented with 2% of antibiotic and nonessential amino acids. (FIG. 1).

[0170] ii. Culture Conditions: [0171] nutritive culture medium A for fibroblast amplification:

[0172] The nutritive culture medium A for fibroblast amplification has the following composition:

TABLE-US-00001 Final concentrations Volumes DMEM Glutamax 78% by volume 390 ml Gibco No. 31966047 Fetal calf serum (FCS) 20% by volume 100 ml FetalClone No. SH30072.03 Nonessential amino acids 1% by volume 5 ml Gibco No. 11140-035 Antibiotics/Antimycotics 1% by volume 5 ml Gibco No. 15240-062

[0173] Details of the commercial media used for the preparation of the culture medium A

[0174] DMEM Glutamax medium (Gibco No. 31966047)

TABLE-US-00002 Compounds Concentration (mg/l) Glycine 30.0 L-Alanyl-L-Glutamine 862.0 L-Arginine hydrochloride 84.0 L-Cystine dihydrochloride 63.0 L-Histidine hydrochloride hydrate 42.0 L-Isoleucine 105.0 L-Leucine 105.0 L-Lysine hydrochloride 146.0 L- Methionine 30.0 L-Phenylalanine 66.0 L-Serine 42.0 L-Threonine 95.0 L-Tryptophan 16.0 L-Tyrosine 72.0 L-Valine 94.0 Choline chloride 4.0 Calcium D-pantothenate 4.0 Folic acid 4.0 Niacinamide 4.0 Pyridoxine hydrochloride 4.0 Riboflavin 0.4 Thiamine hydrochloride 4.0 i-Inositol 7.2 Calcium chloride (CaCl22H2O) 264.0 Iron nitrate (Fe(NO3)39H2O) 0.1 Magnesium sulfate (MgSO47H2O) 200.0 Potassium chloride (KCl) 400.0 Sodium bicarbonate (NaHCO3) 3700.0 Sodium chloride (NaCl) 6400.0 Sodium dihydrogen phosphate 141.0 (NaH2PO42H2O) D-Glucose (Dextrose) 4500.0 Phenol Red 15.0

[0175] Nonessential amino acids Gibco No. 11140-035

TABLE-US-00003 Compounds Concentration (mg/l) Glycine 750.0 L-Alanine 890.0 L-Asparagine 1320.0 L-Aspartic acid 1330.0 L-Glutamic acid 1470.0 L-Proline 1150.0 L-Serine 1050.0

[0176] Antibiotics/Antimycotics Gibco No. 15240-062

TABLE-US-00004 Compounds Concentration (mg/l) Penicillin 10 000 U/ml Streptomycin 10 000 g/ml Amphotericin b 25 g/ml [0177] nutritive culture medium B for preparing the dermal papilla equivalents

[0178] The nutritive culture medium B for preparing the dermal papilla equivalents has the following composition:

TABLE-US-00005 Final concentrations Volumes Williams E (glutamine-free) 98% by volume 489 ml Gibco No. A12176-01 L-glutamine 2 mM 5 ml Antibiotics/Antimycotics 1% by volume 5 ml Gibco No. 15240-062 Insulin 10 g/ml 500 l Hydrocortisone 10 ng/ml 10 l

[0179] Details of the commercial media used for the preparation of the culture medium B

[0180] Williams E medium (glutamine-free) (Gibco No. A12176-01)

TABLE-US-00006 Compounds Concentration (mg/l) Glycine 50.0 L-Alanine 90.0 L-Arginine 50.0 L-Asparagine hydrate 20.0 L-Aspartic acid 30.0 L-Cysteine 40.0 L-Cystine dihydrochloride 26.07 L-Glutamic acid 50.0 L-Histidine 15.0 L-Isoleucine 50.0 L-Leucine 75.0 L-Lysine hydrochloride 87.46 L-Methionine 15.0 L-Phenylalanine 25.0 L-Proline 30.0 L-Serine 10.0 L-Threonine 40.0 L-Tryptophan 10.0 Disodium salt of L-tyrosine 50.65 dihydrate L-Valine 50.0 Ascorbic Acid 2.0 Biotin 0.5 Choline chloride 1.5 Calcium D-pantothenate 1.0 Ergocalciferol 0.1 Folic acid 1.0 Menadione sodium bisulfate 0.01 Niacinamide 1.0 Pyridoxal hydrochloride 1.0 Riboflavin 0.1 Thiamine hydrochloride 1.0 Vitamin A (acetate) 0.1 Vitamin B12 0.2 alphaTocopherol phos. 0.01 sodium salt i-Inositol 2.0 Calcium chloride (CaCl2) (anhydrous) 200.0 Copper sulfate (CuSO45H2O) 1.0E4 Iron sulfate (FeSO47H2O) 1.0E4 Magnesium sulfate (MgSO4) (anhydrous) 97.67 Manganese sulfate (MnSO4H2O) 1.0E4 Potassium chloride (KCl) 400.0 Sodium bicarbonate (NaHCO3) 2200.0 Sodium chloride (NaCl) 6800.0 Anhydrous sodium dihydrogen 140.0 phosphate (NaH2PO4) Zinc sulfate (ZnSO47H2O) 2.0E4 D-Glucose (Dextrose) 2000.0 Glutathione (reduced) 0.05 Methyl linoleate 0.03 Sodium pyruvate 25.0

[0181] Antibiotics/Antimycotics Gibco No. 15240-062 (cf. above)

Culture-Amplification of the Dermal Papilla Fibroblasts

[0182] The dermal papilla located in the bulb region of the follicle is pinpointed under the microscope. The dermal papilla is microdissected using a scalpel and needles and is then placed in a culture dish containing the nutritive culture medium A as described above. (FIG. 3).

Culture-Preparation of the Dermal Papilla Equivalent

[0183] After amplification of the fibroblasts in monolayer culture, the fibroblasts are trypsinized and then deposited in a Petri dish not treated for cell culture (Falcon bacteriological Petri dish, Corning, ref.: 351007) at high density (for example 23 800 cells per cm.sup.2), in the serum-free nutritive culture medium B as described above.

[0184] The fibroblasts migrate in the Petri dish and group together to form clusters, then cell aggregates, and finally detach from the support so as to form dermal papilla spheroids or equivalents after 5 days of culture. (FIGS. 4 and 12).

[0185] The dermal papillae obtained are in spherical form with a diameter of about 200 m.

[0186] Labeling of the alkaline phosphatase enzymatic activity is performed using the NBT/BCIP alkaline phosphatase kit (Roche ref.: 11 681 451) in which BCIP (5-bromo-4-chloro-3-indolyl phosphate, toluidine salt), the alkaline phosphatase substrate, will first be dephosphorylated then oxidized to give a blue-colored product.

[0187] A vivid dark violet color shows a strongly positive alkaline phosphatase enzymatic activity. (FIG. 8).

[0188] Dermal papilla equivalents according to the invention obtained according to the same process as that of Example 1 were also prepared, replacing: [0189] the 2D support (Falcon bacteriological Petri dish, Corning) with the round-bottomed 96-well 3D microplate support sold under the name Costar by the company Corning; [0190] the fibroblast seeding density of 23 800 cells/cm.sup.2 with 9375 cells/cm.sup.2.

[0191] The dermal papillae obtained are also of spherical shape with a diameter of about 200 m (see FIG. 15) and have a highly positive alkaline phosphatase enzymatic activity.

[0192] Conclusion: The dermal papillae according to the invention thus obtained do indeed have the morphological and functional features of an in vivo dermal papilla.

EXAMPLE 2PREPARATION OF A HAIR FOLLICLE EQUIVALENT ACCORDING TO THE INVENTION

Experimental Protocol

[0193] i. Microdissection of the Matrix Cells

[0194] The hair follicles are extracted from a surgical residue of scalp. Said residue is first cut into 5 mm.sup.2 portions and then sectioned using a scalpel between the dermis and the hypodermis.

[0195] The follicles are extracted using ophthalmic surgery forceps and are then sectioned just above the papilla with a scalpel. The bulb is then recovered. At this stage, the bulb comprises two compartments: the dermal compartment (dermal papilla and connective tissue sheath) and the matrix cells which form a cell mass. (FIG. 1).

[0196] The epithelial part is separated from the dermal part using perfusion needles.

[0197] ii. Culture Conditions

[0198] The culture conditions have three main components: [0199] The base medium:

[0200] Unless otherwise indicated, all of the media and buffers used in the examples are described in Bell et al. 1979, (P.N.A.S. USA, 76, 1274-1278), Asselineau and Prunieras, 1984, (British J. of Derm., 111, 219-222) or Asselineau et al., 1987, (Models in dermato., vol. III, Ed. Lowe & Maibach, 1-7).

[0201] The DMEM medium+10% FCS+7F (called G7F medium) has the following composition:

TABLE-US-00007 Final concentrations DMEM 500 ml Fetal calf serum (FCS) 10% L-Glutamine 2 mM Sodium pyruvate 1 mM Penicillin - Streptomycin Penicillin 20 U/ml Streptomycin 20 g/ml Fungizone Penicillin 10 U/ml Streptomycin 10 g/ml Amphotericin-B 25 ng/ml Epidermal growth factor (EGF) 10 ng/ml Cholera toxin 10.sup.10 M Hydrocortisone 0.4 g/ml Adenine hydrochloride 1.8 10.sup.4 M Triiodothyronine (T3) 2 10.sup.9 M Human transferrin 5 g/ml Bovine insulin 5 g/ml [0202] The culture supplements: 10 M of Y27632. [0203] The adhesion surface: the matrix cells adhere and proliferate in the Green base medium in the presence of a feeder layer of murine 3T3 fibroblasts arrested in the cell cycle by mitomycin treatment.

Culture-Amplification of the Matrix Cells

[0204] After microdissection, the matrix cell clumps are deposited in Petri dishes 60 mm in diameter, seeded beforehand with a feeder layer, preferably with 40 000 irradiated 3T3 cells/cm.sup.2, and covered with the complete culture medium, for example the G7F medium+10 M Y27632; a culture of matrix cells at 70% confluence is obtained.

Culture-Preparation of the Hair Follicle Equivalent

[0205] In order to generate the in vitro hair follicle equivalents, the cells are recovered at the subconfluent stage by enzymatic treatment. The cells are then seeded in bacteriological Petri dishes not treated for cell culture (Falcon bacteriological Petri dish, Corning, ref.: 351007), containing beforehand the dermal papilla equivalents, at a density of 6000 cells/cm.sup.2, in the serum-free nutritive culture medium B as described in Example 1.

[0206] The matrix cells adhere only at the level of the dermal papilla spheroids and proliferate. (FIGS. 5 and 6).

[0207] After 6 days of coculture, hair follicle equivalents are seen to appear, in particular having the following morphological features: solid tubular structure about 2500 micrometers in diameter. (FIGS. 6 and 7).

[0208] The labeling of the hair-specific keratins K85 and K35, and also a cell proliferation marker, such as Ki67, is performed by fluorescence immunolabeling. (FIG. 9).

[0209] Conclusion: The hair follicles according to the invention thus obtained do indeed have the morphological and functional features of an in vivo hair follicle.

EXAMPLE 3

Comparative Outside the Invention: Dermal Papilla Obtained According to the Process Described in Example 2 of WO 2009/118283

[0210] 1) Materials and Methods:

[0211] The preparation of the dermal papilla equivalent was performed according to the preparation protocol described in example 2 of WO 2009/118283.

[0212] Preparation of the DMEM (+) medium: [0213] 500 ml of DMEM Glutamax (Invitrogen No. 31966) [0214] 5 ml of nonessential amino acids (NEAA) (Gibco No. 11140-035) [0215] 50 l of ascorbic acid at 100 mM (Sigma No. A8960), i.e. 2.9 mg/l final [0216] 5 ml of Insulin-Transferrin-Sodium Selenite (ITS) (Fisher Scientific No. 10524233) [0217] 400 mg/l BSA

[0218] Culture support: Flat-bottomed 6-well ULA (ultra low attachment) 3D plate.

[0219] Cell density: 6660 cells/cm.sup.2.

[0220] Cells: fibroblasts obtained from a dermal papilla (passage P6).

[0221] 2) Results: (See FIG. 11)

[0222] Conclusion: very heterogeneous cell aggregates of different sizes with irregular edges are observed.

EXAMPLE 4

Comparative Outside the Invention: Dermal Papilla Obtained According to the Process Described in Example 3 of WO 2009/118283

[0223] 1) Materials and Methods:

[0224] The preparation of the hair follicle equivalent was performed according to the preparation protocol described in example 3 of WO 2009/118283.

[0225] Culture Medium: [0226] DMEM (+) for the papilla equivalents (DMEM+2% BSA+ITS+Vit C) [0227] Then KSFM for the DP/MHN/ORS mixed culture

[0228] Culture support: Flat-bottomed 6-well ULA (ultra low attachment) plate.

[0229] Cell Density: [0230] For the preparation of the dermal papilla: fibroblasts obtained from a dermal papilla: 500 000 DP/F75 i.e. 6660 DP/cm.sup.2; [0231] For the preparation of the hair follicle: 250 000 ORS/6 wells, i.e. 26 300 ORS/cm.sup.2+25 000 MHN/6 wells, i.e. 2630 MHN/cm.sup.2.

[0232] Cells: [0233] Fibroblasts obtained from a dermal papilla (passage P6). [0234] Melanocytes M597 (passage P4) [0235] Keratinocytes from ORS IB (passage P3)

[0236] 2) Results (See FIG. 13)

[0237] Conclusion: cell aggregates (cysts) are observed without evolution toward a hair follicle.

EXAMPLE 5

Comparative Outside the Invention: Culture of Fibroblasts Obtained from Dermal Papilla on a 2D Culture Support for Cell Culturing (i.e. Support Permitting Cell Adhesion)

[0238] 1) Materials and Methods:

[0239] The preparation of the dermal papilla equivalent was performed according to the preparation protocol described in example 1, replacing the Falcon bacteriological Petri dish culture support, Corning, ref.: 351007 with a Petri dish culture support for cell culturing (i.e. permitting cell adhesion).

[0240] 2) Results: (See FIG. 14)

[0241] Conclusion: No formation of dermal papilla is observed using a culture support permitting cell adhesion.

EXAMPLE 6

Comparative Outside the Invention: Dermal Papilla Obtained in a Culture Medium Containing Serum as Described in Higgins et al. (Modelling the Hair Follicle Dermal Papilla Using Spheroid Cell Cultures)

[0242] 1) Materials and Methods:

[0243] Comparative Outside the Invention: [0244] 3D Culture support: U-bottomed 96-well ULA plate [0245] Culture medium: DMEM medium+10% FCS [0246] Cell density: 9375 cells/cm.sup.2

[0247] Dermal Papilla Equivalent According to the Invention: [0248] 3D Culture support: U-bottomed 96-well ULA plate [0249] Culture medium: Williams medium (serum-free) [0250] Cell density: 9375 cells/cm.sup.2

[0251] In order to measure the RNA expression of the alkaline phosphatase, Versican, and SFRP2 secreted frizzled related protein 2, which are markers of the enzymatic activity of the dermal papilla, the probes ALPL-Hs01029144_m1, VCAN-Hs00171642_m1, and SFRP2-Hs00293258_m1 were used.

TABLE-US-00008 Expression variations DP P5 3D DP P5 3D DMEM + 10% WILLIAMS E FCS (outside the (according to the Probes invention) invention) ALPL-Hs01029144_m1 1.00 1.75 VCAN-Hs00171642_m1 1.00 3.61 SFRP2-Hs00293258_m1 1.00 2.18

[0252] Conclusion: [0253] For the dermal papilla equivalent according to the invention, overexpression is observed for the markers ALPL (alkaline phosphatase), VCAN (versican) and SFRP2 (secreted frizzled related protein 2), which are known as markers of inductivity of the enzymatic activity of the dermal papilla. [0254] For the comparative outside the invention, underexpression is observed for the markers ALPL (alkaline phosphatase), VCAN (versican) and SFRP2 (secreted frizzled related protein 2), which are known as markers of inductivity of the enzymatic activity of the dermal papilla.

EXAMPLE 7

Comparative Outside the Invention: Hair Follicle Obtained in 3D Culturing on Collagen Gel

[0255] 1) Materials and Methods:

[0256] A first step of manufacturing of a spheroid comprising fibroblasts obtained from dermal papilla and from proliferative epithelial cells (matrix cells) was performed in DMEM medium+10% serum, in a U-bottomed 96-well ULA plate.

[0257] A second step was then performed, which consists in integrating the spheroids into a collagen gel (lattice), observation being performed on D14.

[0258] 2) Results: (See FIG. 16)

[0259] Conclusion: No formation of hair follicles is observed in the collagen gel; a cyst is observed without apical structure formation.