Reconstructed scalp model and process for screening active molecules

Abstract

The invention relates to a reconstructed scalp model, to the process for preparing it and to its use for evaluating the effect of cosmetic, pharmaceutical or dermatological topical products. The reconstructed scalp according to the invention may also be used for the preparation of the grafts intended for treating cutaneous scalp disorders.

Claims

1. A process for preparing a scalp equivalent, the process comprising seeding and culturing interfollicular scalp keratinocytes on a dermal equivalent, wherein the dermal equivalent comprises collagen and interfollicular scalp fibroblasts, wherein the interfollicular scalp keratinocytes are obtained from a scalp sample from which keratinocytes of an outer epithelial sheath have been removed beforehand.

2. The process of claim 1, wherein the dermal equivalent is a contracted collagen lattice and the seeding with keratinocytes is performed after 2 to 5 days of contraction of the lattice.

3. The process of claim 1, wherein the interfollicular scalp fibroblasts and keratinocytes are isolated, respectively, from dermis and epidermis obtained from a scalp sample.

4. The process of claim 1, wherein the interfollicular scalp keratinocytes are obtained by: separating the scalp dermis from the epidermis by proteolytic treatment of the scalp sample, recovering the scalp epidermis, placing the recovered scalp epidermis in contact with trypsin, recovering the interfollicular scalp keratinocytes, and culturing the recovered keratinocytes.

5. The process of claim 4, wherein the keratinocyte culture is a coculture with fibroblasts treated beforehand with mitomycin or irradiated or a culture on a culture medium supplemented with fibronectin or with collagen.

6. The process of claim 1, wherein the interfollicular scalp fibroblasts are obtained by: separating the scalp dermis from the epidermis by proteolytic treatment of the scalp sample, recovering the scalp dermis, placing the recovered scalp dermis in contact with collagenase, trypsin, or both collagenase and trypsin, recovering the interfollicular scalp fibroblasts, and culturing the recovered fibroblasts.

7. The process of claim 2, wherein the contracted dermal equivalent seeded with the interfollicular scalp keratinocytes is cultured for 5 to 7days by immersion in a culture medium followed by emersion for 5 to 7 days on a suitable support.

8. The process of claim 2, further comprising implanting hair fibroblasts selected from the group consisting of fibroblasts of the dermal papilla, fibroblasts of the connective sheath, whole dermal papillae, connective sheaths, and fractions of connective sheaths into the dermal equivalent that has contracted or that is in the course of contraction.

9. The process of claim 1, wherein the collagen is a type I fibrillar collagen, a type II fibrillar collagen or a type III fibrillar collagen.

10. The process of claim 1, wherein the collagen is a type I fibrillar collagen.

Description

EXAMPLE 1

Preparation of a Scalp Model

(1) Interfollicular scalp keratinocytes and fibroblasts are isolated from scalp samples collected during a face-lift (and thus obtained from relatively mature women) or from scalp samples obtained from relatively young men.

(2) To isolate these scalp cells, a hair epilation is performed so as to remove the keratinocytes of the outer epithelial sheath, and the dermis is then separated from the epidermis in order to extract the two target cell populations, namely the interfollicular fibroblasts and keratinocytes.

(3) Experimental Protocol:

(4) Unless otherwise indicated, all 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 and Maibach, 1-7).

(5) The medium MEM+10% FCS+3F (known as 3F medium) has the following composition:

(6) TABLE-US-00001 Reagent Brand Volume MEM Biochrom KG 500 ml L-Glutamine 200 mM Gibco 5 ml Sodium pyruvate 100 mM Biochrom KG 5 ml NEA Biochrom KG 5 ml FCS Biochrom KG 50 ml Penicillin-streptomycin Biochrom KG 1 ml Antibiotic-antimycotic Gibco 0.5 ml EGF 10 g/ml TEBU 0.5 ml Cholera toxin 10.sup.5 M Sigma 5 l Hydrocortisone 0.5 mg/ml Sigma 0.4 ml Place the pieces of Top part in a culture dish of diameter 100 mm containing 30-40 ml of dispase (Roche) and store overnight (about 15 hours) at 4 C. or store for 1 hour 30 minutes at 37 C. in a dry incubator, care being taken to cut small pieces.
1. Preparation of the Primary Culture of Normal Human Keratinocytes Remove the samples from the dispase bath and place them in a culture dish (diameter 100 mm) containing 10 ml of trypsin-EDTA (0.05%-0.02%). Perform the separation of the dermis from the epidermis using curved tweezers and gently scrape the surface of the dermis with the back of the tweezers so as to recover the basal keratinocytes. Place the epidermis in a conical tube (50 ml) with the 10 ml of trypsin-EDTA from the dish. Rinse the dish with 5 ml of trypsin-EDTA. Filter through sterile gauze. Neutralize with 20 ml of pure foetal calf serum (FCS). Place the suspension in a 50 ml conical tube. Centrifuge the cell suspension for 10 minutes at 1200 rpm. Remove the supernatant, take up the pellet with MEM 10% FCS+7F (this medium comprises the same compounds as the 3F medium and four additional compounds: adenine, transferrin, T3, insulin). Count the viable cells. Seed the culture dishes with 10 000 live cells/cm.sup.2. 24 hours later, perform a standard coculture by adding the 3T3 (12 000 cells/cm.sup.2) treated with mitomycin C (Ametycine) without changing the medium.
2. Protocol for Isolating the Dermal Cells: a) Prepare a solution comprising 0.1% glucose, 0.8% NaCl and 0.04% KCl. Add collagenase to 0.1% (Worthington). Filter the solution with a Millipore or Nalgene 0.22 m filter. b) Place the dermis in the form of small pieces with a side length of about 2 mm, 20 ml of the collagenase solution and a sterile bar in a 50 ml beaker. Stir at 37 C. under 5% CO.sub.2 for 1 hour 30 minutes. Filter the suspension obtained through a double thickness of sterile gauze. Centrifuge the filtered suspension under the usual conditions for cell centrifugation. c) Resuspend the pellet in fresh culture medium, count the cells and seed the dishes.
3. Preparation of the Lattices:

(7) If the experiment to be performed comprises more than 25 lattices, all the above volumes must be doubled.

(8) MEM 1.76X

(9) 17.6 ml of MEM 10 5.1 ml of NaHCO.sub.3 7.5% 0.88 ml of L-Glutamine (1.76 mM) 0.88 ml of sodium pyruvate (0.88 mM) 0.88 ml of non-essential amino acids 0.88 X (Ref.: K 0293Supplier: Biochrom KG) 0.088 ml of penicillin streptomycin 8.8 U/8.8 g/ml (0.088%) (Ref.: A2212Supplier: Biochrom KG) 0.044 ml of antimycotic antibiotic 0.04 X (0.04%) 75 ml of sterile ultrapure water
MEM HEPES 10% FCS 50 ml of MEM 25 mM HEPES 0.5 ml of L-Glutamine (2 mM) 0.5 ml of sodium pyruvate (1 mM) 0.5 ml of non-essential amino acids 1 X 0.1 ml of penicillin streptomycin 20 U/20 g/ml (0.2%) 0.05 ml of antimycotic antibiotic 0.1 X (0.1%) 5 ml of FCS 10%.
NaOH 0.1N 10 ml NaOH 1N 90 ml of sterile ultrapure water

(10) This solution is passed through a Millipore filter with a GV Durapore 0.22 m membrane.

(11) Acetic acid 1/1000

(12) 0.5 ml of 100% glacial acetic acid 499.5 ml of sterile ultrapure water
Using the media thus prepared: Prepare in a sterile conical flask 5 ml of the following solution: 3.22 ml of MEM 1.76 X 0.63 ml of FCS 0.35 ml of NaOH 0.1 N 0.6 ml of acetic acid 1/1000 0.2 ml of MEM HEPES 10% FCS. Add 0.5 ml of cell suspension of interfollicular scalp fibroblasts to this solution. Next, add slowly the necessary volume of cold collagen (2.1 ml if this collagen is Gattefoss or Symatse collagen or 1.5 ml if it is Coletica collagen). Shake the conical flask vigorously until homogenized (the fuchsia-pink solution turns salmon coloured). Empty the contents of the conical flask into a 60 mm Falcon bacteria dish. Place the dish in the oven (37 C.-5% CO.sub.2) for about 1 hour 30 minutes-2 hours. When the gel has set and the medium expelled, check the start of contraction. In the afternoon, shake the lattices regularly so that they do not stick together again. Leave the contraction to proceed for 3 days.
4. Culture-seeding with Keratinocytes

(13) The seeding with keratinocytes is performed after 3 days of contraction of the lattices.

(14) a) Preparation of the Adhesive

(15) Prepare 0.7 ml of the following solution, given to bond two lattices: 0.46 ml of MEM 1.76 X 0.09 ml of FCS 0.05 ml of NaOH 0.1 N 0.1 ml of MEM HEPES 10% FCS. Add to this tube 0.3 ml of dialysed collagen. The final volume is thus 1 ml.
Comments: The above volumes are given for the bonding of two lattices. For several lattices, prepare a global solution corresponding to n+1 samples to be bonded in a conical flask. Dispense, by tube, 0.7 ml of this solution using a multipipette. Next, add 0.3 ml per tube of collagen. Work the lattices two by two. b) Bonding of the Lattices Take a 6 ml tube filled with adhesive solution. Stir with a vortex mixer. Take up the solution by pipette and place in two Corning culture dishes a drop of about 0.45 ml of this solution into the middle of each dish. Take up a lattice using curved tweezers and a cell lifter. Place the lattice in the lid of its original dish in order to remove the surplus medium. Take up the lattice and place it on the drop of adhesive. Spread uniformly by rotating the dish so that the adhesive is distributed around the lattice. Place the dishes in an oven (37 C.-5% CO.sub.2) for 20-30 minutes to allow the adhesive to set. c) Seeding with Keratinocytes Thaw the cells as quickly as possible by stirring the vial in a water bath at 37 C., if these cells were frozen. Transfer the contents of the vial into a 50 ml Falcon culture tube containing 35 ml of MEM 10% FCS+3F medium. Centrifuge for 5 minutes at 1000 rpm. Remove the supernatant. Take up the centrifugation pellet with MEM 10% FCS+3F medium to obtain a solution containing 100 000 cells/ml. Resuspend the cells by suction-ejection several times. Place a seeding ring 14 mm in diameter on the bonded lattices. In this ring, place 0.5 ml of cell suspension (=50 000 cells/ring 1.5 cm.sup.2, i.e. about 33 000 cells per cm.sup.2). Around the ring, add 5 to 7 ml of MEM 10% FCS+3F medium gently so as not to detach the lattice. In parallel, seed two control rings (dish with rings not containing lattice). Place the dishes in an oven at 37 C.-5% CO.sub.2 for 2 hours. Remove the rings using curved tweezers after the 2 hours of attachment. Return the dishes to the oven at 37 C.-5% CO.sub.2. Change the medium on Wednesday and Friday (5 to 7 ml of MEM 10%FCS+3F medium per dish).
Culture-Emersion of Reconstructed Skins

(16) After seven days of culturing in immersion, the skins are placed in emersion.

(17) Check, however, that the keratinocytes emerge from the lattice by observing with a microscope. Place an emersion grille in a Falcon bacteria dish. Add 7.5 ml of MEM 10% FCS+3 F while taking care to avoid the formation of bubbles. Cut the adhesive around the skin to be emerged using a sterile scalpel. Transfer the skin onto the grille with curved tweezers and a cell lifter. Place the dishes in the oven at 37 C.-5% CO.sub.2. Change the medium on Wednesday and Friday (7 to 7.5 ml of MEM 10%FCS +3F medium).

(18) After seven days of emersion, the reconstructed scalp skins are ready to be used.

EXAMPLE 2

Comparison of the Expression of Involucrin and of Keratin K10

(19) The expression of involucrin and of keratin K10 in the scalp equivalent obtained in Example 1 is observed after immunolabelling using mouse monoclonal antibodies directed against involucrin or against K10. This expression is also observed in a standard skin equivalent and in a scalp sample.

(20) Preparation of the Standard Skin Equivalent and of the Scalp Sample:

(21) To prepare a standard dermal equivalent, 3.22 ml of MEM 1.76 X medium, 0.63 ml of foetal calf serum, 0.35 ml of 0.1 N sodium hydroxide and 0.20 ml of an MEM/HEPES medium mixture containing 10% foetal calf serum (MEM/HEPES/FCS10) are placed in a sterile Falcon tube.

(22) 0.50 ml of MEM/HEPES/FCS10 medium containing fibroblasts obtained from human mammary surgeries prepared beforehand according to the method described by Bell et al. 1979 (P.N.A.S. USA, 76, 1274-1278), Asselineau and

(23) Prunieras, 1984, (British J. of Derm., 111, 219-222) or Asselineau et al., 1987, (Models in dermato., vol. III, Ed. Lowe & Maibach, 1-7), are then added to a concentration of 110.sup.6 cells per 0.5 ml of culture medium.

(24) 2 ml of a volume/volume mixture of collagen at a concentration of 3 mg/ml in acetic acid to 1/1000 are then added slowly, down the wall of the tube so as to observe the appearance of a whitish cloud. The whole is then mixed cautiously and placed in a Petri dish 60 mm in diameter (Falcon 60 mm type, ref. 1016). The Petri dish is then placed in an oven at 37 C. and left for about 2 hours 30 minutes. When the appearance of two phases (gel+medium) is observed, the lattice is cautiously detached from its support, and the lattice thus detached from its support is left for 4 days in the oven.

(25) To prepare a standard skin equivalent, the standard dermal equivalent is spread in a Corning type culture dish 60 mm in diameter on a drop of collagen adhesive (0.6 ml) and then maintained at 37 C. in an oven for 20-30 minutes.

(26) A sterile steel ring is placed on the lattice and 0.5 ml of a cell suspension of human keratinocytes obtained from mammary surgeries prepared according to Rgnier et al. (Frontier of Matrix Biology, Vol. 9, 4-35, Karger, Basle 1981), at a rate of 100 000 cells/ml in MEM 10% FCS+3F medium are placed inside the ring.

(27) About 6 ml of medium (MEM 10% FCS+3F) are placed around the ring and the dish is placed in an oven at 37 C. for 2 hours. The ring is then removed and the dish is returned to the oven.

(28) After eight days, the culture is then placed at the air/liquid interface, the said liquid then consisting of the same medium as previously.

(29) Culturing is then continued for 1 week until a histologically satisfactory epidermal equivalent is obtained, i.e. an epidermal equivalent that has the four standard cell layers, namely the basal, suprabasal, granular and horny layers.

(30) The scalp sample is recovered after lifting according to the methods known to those skilled in the art.

(31) Measurement of the Expression of Involucrin and of Keratin K10

(32) After freezing, the various tissues are cut into slices 5 m thick using a cryostat.

(33) The slices are then rinsed twice with PBS and 25 l of anti-involucrin antibody (Sigma - Ref.: I9018) and anti K10 antibody (Immuquest Ltd -Ref. AE20) diluted to 1/50 are placed on each slice and left for 30 minutes at room temperature (25 C.). The slices are then rinsed twice with PBS and 25 l of FITC conjugated antibody (Rabbit anti-mouse FITC, Dako F232), are deposited on each slice and left for 30 minutes at room temperature (25 C.). The slices are rinsed twice with PBS and observed after mounting under a Leica brand fluorescence microscope, Leitz DMRB model.

(34) The observation shows that in the control (reconstructed skin), involucrin (FIG. 1, I) and keratin K10 (FIG. 1, II) are expressed in all the suprabasal layers of the reconstructed epidermis (FIG. 1, A), whereas they are expressed tardively, i.e. only in the granular layers, of the epidermis of the scalp equivalents (FIG. 1, B) and of the scalp samples (FIG. 1, C).

(35) The expression of these two markers takes place early in the reconstructed skin (A) and tardively in the reconstructed scalp model (B) and in the scalp sample (C) at the places indicated by the arrows in FIG. 1.

(36) These results thus make it possible to confirm that the reconstructed scalp model according to the invention is similar to the structure and functionality of an in vivo scalp.