Use of matrix cells for preparing a micro hair follicle

Abstract

The invention relates to the use of matrix cells for obtaining a micro hair follicle and to the use thereof for evaluating the effect of cosmetic, pharmaceutical or dermatological products and also for the prophylactic or therapeutic treatment of a state of reduced pilosity.

Claims

1. One or more micro hair follicles in the form of a bud that is obtained by means of an in vitro process comprising at least one step of culturing matrix cells which are located in the hair bulb in the presence of an effective amount of a ROCK inhibitor for a period of time sufficient to allow differentiation of said cells into keratinocytes positive for the K85 K35 markers, wherein the cells are keratinocytes positive for the K85 K35 markers and upon reaching confluence are in the form regular clusters and wherein the matrix cells are obtained from hair in the anagen phase and are free from other cell types, and wherein at least one bud comprises a structure in the form of keratinized fibre.

2. The one or more micro hair follicles according to claim 1, in which the ROCK inhibitor is Y27632.

3. The one or more micro hair follicles according to claim 1, in which the effective amount of the ROCK inhibitor is from 1 to 100 μM.

4. The one or more micro hair follicles according to claim 1, in which the effective amount of the ROCK inhibitor is from 5 to 25 μM.

5. The one or more micro hair follicles according to claim 1, in which the effective amount of the ROCK inhibitor is 10 μM.

6. The one or more micro hair follicles according to claim 1, in which the matrix cells are cultured in the presence of the ROCK inhibitor for at least 2 days.

7. The one or more micro hair follicles according to claim 1 wherein said process comprises the following steps: a—isolating a follicle on a support; b—cutting, on said support immersed in a culture medium, the bulb region above the dermal papilla; c—recovering the matrix cells in the form of a cell aggregate on said support; d—amplifying the matrix cells in the presence of a ROCK inhibitor; e—recovering keratinocytes positive for K35 K85 markers; f—culturing the keratinocytes obtained in step e) in 3D culture.

8. The one or more micro hair follicles according to claim 7, wherein in step f), the number of keratinocytes placed in culture is between 1000 and 4000 cells.

9. The one or more micro hair follicles according to claim 1, which are constituted of cells resulting from amplification and differentiation of matrix cells which are located in the hair bulb in the presence of a ROCK inhibitor.

10. The one or more micro hair follicles according to claim 9, wherein the ROCK inhibitor is Y27632.

11. A process for a prophylactic or therapeutic treatment of a state of reduced pilosity, which comprises implanting the one or more micro hair follicles according to claim 1, into a subject in need of the prophylactic or therapeutic treatment of a state of reduced pilosity.

12. A process for treating alopecia which comprises implanting the one or more micro hair follicles according to claim 1, into a subject having alopecia.

13. A process for the in vitro testing of effects of an active agent on hair properties which comprises contacting the active agent with the one or more micro hair follicles as described in claim 1.

14. The process according to claim 13 further comprises identifying a compound which modulates the growth of body hair and/or of head hair.

15. A product screening process for the purpose of identifying novel active agents, comprising a step (a) of bringing a test product into contact with the one or more micro hair follicles according to claim 1, then a step (b) of analyzing the effect of said product on at least one parameter of a scalp equivalent and a step (c) of selecting the product which modifies said parameter.

16. The one or more micro hair follicles according to claim 2, in which the effective amount of the ROCK inhibitor is from 1 to 100 μM.

Description

DESCRIPTION OF THE FIGURES

(1) The figures make it possible to give a better illustration of the invention, without however limiting the scope thereof.

(2) FIG. 1: Matrix cell 1 localization

(3) FIG. 2: Section above the dermal papilla

(4) FIG. 3: Bulb containing the matrix cells

(5) FIG. 4: Matrix cell clump deposited on a 3T3 feeder layer

(6) FIG. 5: Matrix cells after 3d of culture

(7) FIG. 6: Matrix cells at confluence after 10 days of culture

(8) FIG. 7: Primary culture of matrix cells with cluster formation

(9) FIG. 8: Sphere of matrix cells after 3d of 3D culture

(10) FIG. 9: Bud formation in 3D culture

(11) FIG. 10: Buds are strongly positive for K85 and K35

(12) FIG. 11: spheres exhibiting a micro hair follicle

(13) FIG. 12: No bud formation in 3D culture from ORS keratinocytes

(14) The examples given below are presented as non-limiting illustrations of the invention.

EXAMPLE 1—PREPARATION OF A MICRO HAIR FOLLICLE

(15) Experimental Protocol

(16) i. Matrix Cell Microdissection

(17) 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.

(18) The follicles are extracted using ophthalmic surgery forceps and are then sectioned just above the papilla with a scalpel (FIG. 2). The bulb is then recovered (FIG. 3). At this stage, the bulb comprises two compartments: The dermal compartment (dermal papilla and connective tissue sheath). The matrix cells which form a cell mass.
The epithelial part is separated from the dermal part using perfusion needles.

(19) ii. Culture Conditions:

(20) The culture conditions have three main components:

(21) The base medium:
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).
The MEM medium+10% FCS+3F (called 3F medium) has the following composition:

(22) TABLE-US-00001 MEM Final concentrations Foetal calf serum (FCS) 10% L-Glutamine 2 mM Sodium pyruvate 1 mM Penicillin - Streptomycin Penicillin 20 U/ml Streptomycin 20 μg/ml Fungizone Penicillin 10 U/ml Streptomycin 10 μg/ml Amphotericin-B 25 ng/ml Epidermal growth factor (EGF) 10 ng/ml Cholera toxin 10.sup.−10 M Hydrocortisone 0.4 μg/ml Adenine hydrochloride 1.8 × 10.sup.−4 M Triiodothyonine (T3) 2 × 10.sup.−9 M Human transferrin 5 μg/ml Bovine insulin 5 μg/ml the culture supplements: growth factors which signal to the cell that it must divide or differentiate. 10 μM of Y27632. the adhesion surface: the matrix cells adhere and proliferate in the Green-based medium in the presence of a feeder layer of murine 3T3 fibroblasts arrested in the cell cycle by mitomycin treatment.
Culture-Amplification of Keratinocytes
After microdissection, the matrix cell clumps are deposited in Petri dishes 60 mm in diameter, seeded beforehand with 1 million 3T3 cells, and covered with the complete culture medium; a culture of matrix cells at confluence is obtained.
In order to generate the spheres, the cells are recovered at the subconfluent stage by enzymatic treatment. The spheres can be obtained in various ways with the conventional techniques already described (hanging drop, centrifugation, non-adhesive support). In the case of the hanging drop, 3000 cells are placed in the plates in sphero. They are recovered in 96-well plates after 48 h in order to monitor their progression.

(23) The non-dissociated matrix cell clumps are isolated and deposited on a 3T3 feeder layer (FIG. 4). After 3d of culture, the cells begin to proliferate in the form of a colony around the matrix clump (FIG. 5). The cells reach subconfluence in 10 days (FIG. 6). They are characterized by a very small size and a strong proliferative capacity. In order to ensure amplification, the cells are then seeded into a T75 flask. Thus, after 3 weeks of culture of 3 bulbs, it is possible to generate approximately 40 million matrix cells at passage 1.

(24) It was possible to observe that the matrix cells were capable of very rapidly forming cell aggregates in vitro. This phenomenon appears to be due to the taxis properties of these cells. In the green 7F medium, these clumps form a regular pattern which recalls that of the follicle buds (FIG. 7).

(25) Furthermore, during the formation of these clumps, the production of numerous spherical cells in suspension occurs, and said cells can be subcultured to generate new cell cultures. After characterization, the buds represent a 1st phase of differentiation of the matrix cells and they are positive for the early keratins of the hair: K35 and K85.

(26) 3D culture with the matrix cells was also carried out. These cells form spheres after 3d of culture (FIG. 8).

(27) After some 7 days of culture, these spheres change conformation, showing a polarized organization. A budding appears, which seems to indicate differentiation of the spheres (FIG. 9). These buds are strongly positive for K85 and K35.

(28) Some spheres exhibit a structure in the form of a fibre which appears to be keratinized (FIG. 10) and which is not found starting from an ORS keratinocyte culture.