SKIN EQUIVALENT AND USE

Abstract

Some embodiments are directed to an in vitro skin, in particular animal skin, including, mammalian and/or human skin, equivalent, and to the use thereof. In particular, the subject matter of some embodiments includes the use of a skin equivalent as a laboratory tool and/or in a method for testing cosmetic and/or dermatological compounds.

Claims

1. An in vitro skin equivalent which can be obtained by a method comprising the steps of: a. culturing of fibroblasts in a fibroblast culture medium M1; b. seeding of a collagen matrix with fibroblasts resulting from step a; c. culturing of fibroblasts seeded in the collagen matrix in a fibroblast culture medium M2 comprising ascorbic acid or an ascorbate or a derivative thereof, the matrix and the cultured fibroblasts forming a dermal substitute; d. culturing of melanocytes in a melanocyte culture medium M3; e. culturing of keratinocytes in a keratinocyte culture medium M4; f. mixing of melanocytes obtained in step d with keratinocytes obtained in step e; g. seeding of the dermal substitute obtained in step c with the mixture obtained in step f; and h. culturing of the dermal substitute seeded in step g in a skin culture medium M5 thus forming the skin substitute.

2. The skin equivalent as claimed in claim 1, wherein the medium M2 includes ascorbic acid.

3. The skin equivalent as claimed in claim 1, wherein the medium M5 includes hyaluronic acid or a hyaluronate or a derivative thereof.

4. The skin equivalent as claimed in claim 3, wherein the medium M5 includes ascorbic acid or an ascorbate or a derivative thereof.

5. The skin equivalent as claimed in claim 1, wherein the mixing of melanocytes and keratinocytes of step f is carried out with a melanocytes/keratinocytes ratio of 1/20 to 1/15.

6. The skin equivalent as claimed in claim 1, wherein the seeding in step g is carried out with a (keratinocytes+melanocytes)/fibroblasts ratio of 9 to 19.

7. The skin equivalent as claimed in claim 1, wherein the seeding in step b is carried out at a density of from 20 000 to 50 000 fibroblasts/cm.sup.2 of surface area of the collagen matrix.

8. The skin equivalent as claimed in claim 1, wherein step c. includes a first culture step for 18 to 28 hours in the presence of a fibroblast culture medium M2.sup.1 including neither ascorbic acid nor ascorbate, followed by a second culturing step for at least two days in the presence of a fibroblast culture medium M2.sup.2 including ascorbic acid or an ascorbate or a derivative thereof.

9. The skin equivalent as claimed in claim 1, wherein step h. further comprises: a first culturing step h. of 18 to 28 hours in the presence of a culture medium M5.sup.1 comprising neither hyaluronic acid, nor hyaluronate, nor ascorbic acid nor ascorbate, a second culturing step h. of at least two days in the presence of a culture medium M5.sup.2 comprising hyaluronic acid or a hyaluronate or a derivative thereof, and a third culturing step h. of at least two days in a medium M5.sup.3 comprising hyaluronic acid or a hyaluronate or a derivative thereof, and ascorbic acid or an ascorbate or a derivative thereof.

10. The use of a skin equivalent as claimed in claim 1 as laboratory tools.

11. The use as claimed in claim 10, wherein the skin equivalent is used in a test method for cosmetic and/or dermatological agents.

12. The use as claimed in claim 11, in which the test method includes the stages of: bringing the skin equivalent into contact with at least one agent, and determining an effect or an absence of effect of said agent.

13. The skin equivalent as claimed in claim 1, wherein the medium M2 includes an ascorbate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0234] FIG. 1 represents a diagram of the steps for obtaining a skin substitute/equivalent.

[0235] FIG. 2 represents optical microscopy photographs of skin (FIG. 2A), and of skin substitutes/equivalents obtained according to the method with variations in the ratio of cells seeded (FIGS. 2B and 2C).

[0236] FIG. 3A is a photograph of a dermal substitute obtained in step c, by optical microscopy after staining of the fibroblasts. FIG. 3B is a photograph of a skin substitute obtained, of small size, namely 0.5 cm.sup.2. FIG. 3C is a photograph of a skin substitute obtained, of medium size, namely 25 cm.sup.2.

[0237] FIG. 4 represents optical microscopy photographs of skin equivalent obtained according to the method with a (keratinocytes+melanocytes)/fibroblasts seeded cell ratio of 13.3 (FIGS. 4A and B); the immunohistochemical labeling of the melanocytes in the basal position (FIG. 4C, light areas) and the production of the basal lamina, labeling of collagen IV (FIG. 4D(2)) and the p63 proliferation marker (FIG. 4D(1)).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0238] A few inventive aspects of the disclosed embodiments are explained in detail below with reference to the various figures. Exemplary embodiments are described to illustrate the disclosed subject matter, not to limit its scope, which is defined by the claims. Those of ordinary skill in the art will recognize a number of equivalent variations of the various features provided in the description that follows.

EXAMPLES

Example 1

Example of Production of a Skin Substitute

[0239] In the present example, the cells used came from a skin biopsy taken from mammoplasties previously carried out on a patient.

[0240] The biopsy was taken by a plastic surgeon and the biopsy was placed in a sterile tube containing physiological saline.

[0241] The cells were isolated from the biopsy as follows:

1. Epithelial Cell Isolation

[0242] a. Rinsing of the biopsy in sterile HBSS (Hank's Balanced Salt Solution). [0243] b. Removal of the adipose tissue by a biologist-technician using a scalpel. [0244] c. Incubation with trypsin-EDTA preheated to 37 C., for between 3 and 24 h. [0245] d. Neutralization with irradiated FCS (trypsin inhibitor). [0246] e. Removal of the epidermis and scraping, with a scalpel, of the basal stratum where the highly proliferative (p63 positive) cells are found. [0247] f. Filtration, centrifugation at 1200 revolutions per minute and seeding of the pellet at 100 000 cells per cm.sup.2 in modified* MCDB153 medium including the compounds mentioned in table 4 below in which the concentrations of L-arginine, L-histidine, L-isoleucine, L-leucine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine and choline chloride have been doubled, the NaCl concentration has been reduced to 0.104 M/l, Hepes has been reduced to 2.29 10.sup.2 M/I and NaHCO.sub.3 has been reduced to 1.19 10.sup.2 M/l, the pH of the medium being adjusted to 7.4 and antibiotics (penicillin and streptomycin 1%) for the keratinocytes.

[0248] For the melanocytes, filtration, centrifugation at 1200 revolutions per minute and seeding of the pellet at 100 000 cells per cm.sup.2 in modified MCDB153 medium including the compounds mentioned in table 4 below, also including 5.88 g of sodium bicarbonate/5I, 0.272 g of tyrosine/5I and 0.157 g of L-methionine/5I, the pH of the medium being adjusted to 7.4.

TABLE-US-00004 TABLE 4 normal composition of the MCDB 153 medium Composition Concentration in g .Math. l.sup.1 Ammonium Metavanadate 0.000000585 Anhydrous calcium chloride 0.00333 Cupric Sulfate5H.sub.2O 0.00000275 Ferrous sulfate7H.sub.2O 0.00139 Magnesium chloride 0.05713 Manganese Sulfate 0.000000151 Molybdic Acid4H.sub.2O (ammonium) 0.00000124 Nickel Chloride6H.sub.2O 0.00000012 Potassium Chloride 0.11183 Sodium Acetate (anhydrous) 0.30153 Sodium Chloride 7.599 Sodium Metasilicate9H.sub.2O 0.000142 Dibasic Sodium Phosphate (anhydrous) 0.284088 Sodium Selenite 0.0000038 Stannous Chloride2H.sub.2O 0.000000113 Zinc Sulfate7H.sub.2O 0.000144 L-Alanine 0.00891 L-ArginineHCl 0.2107 L-AsparagineH.sub.2O 0.015 L-Aspartic Acid 0.00399 L-CysteineHClH.sub.2O 0.04204 L-Glutamic Acid 0.01471 L-Glutamine 0.8772 Glycine 0.00751 L-HistidineHClH.sub.2O 0.01677 L-Isoleucine 0.001968 L-Leucine 0.0656 L-LysineHCl 0.01827 L-Methionine 0.00448 L-Phenylalanine 0.00496 L-Proline 0.03453 L-Serine 0.06306 L-Threonine 0.01191 L-Tryptophan 0.00306 L-Tyrosine2Na 0.00341 L-Valine 0.03513 D-Biotin 0.0000146 Choline Chloride 0.01396 Folic acid 0.00079 myo-Inositol 0.01802 Niacinamide 0.00003663 D-Pantothenic Acid (hemicalcium) 0.000238 PyridoxineHCl 0.00006171 Riboflavin 0.0000376 ThiamineHCl 0.000337 Vitamin B-12 0.000407 AdenineHCl 0.03088 D-Glucose 1.081 HEPES 6.6 Phenol RedNa 0.001242 Putrescine2HCl 0.000161 Pyruvic acidNa 0.055 Thioctic acid 0.000206 Thymidine 0.000727 [0249] g. Incubation at 37 C. at 5% CO.sub.2 for one week with medium changed every three days. [0250] h. After approximately one week: differential trypsinization=trypsinization with 0.025% trypsin and 0.0.1 M EDTA (1-2 minutes in order to detach the melanocytes, 10 minutes in order to detach the keratinocytes). The melanocytes detach first, thereby making it possible to purify the cultures. [0251] Neutralization with irradiated FCS, centrifugation at 1200 revolutions per minute and seeding of the pellet for amplification in the same medium. [0252] i. Incubation at 37 C. at 5% CO.sub.2 for one week with medium changed every three days.

2. Fibroblast Isolation

[0253] a. Rinsing of the dermal part with HBSS. [0254] b. Incubation of the dermis with collagenase at 1% at 37 C. for a maximum of three hours depending on the type of dermis. [0255] c. Neutralization with irradiated FCS. [0256] d. Filtration via a 40 m cell sieve, centrifugation at 1200 revolutions per minute with a GR 2022 centrifuge for 5 minutes and seeding of the pellet at 100 000 cells per cm.sup.2 in DMEM including 10% irradiated FCS and penicillin and streptomycin at 1% for 24 hours. [0257] e. Incubation in a Jouan IG 150 incubator at 37 C., 5% CO.sub.2, for one week with medium changed every three days.

3. Preparation of a Skin Substitute

[0258] a. Trypsinization of the fibroblasts with 0.025% trypsin and 0.0.1M EDTA for 10 minutes, then neutralization with irradiated FCS, centrifugation at 1200 revolutions per minute with a GR 2022 centrifuge for 5 minutes, and seeding in DMEM including 10% FCS on a dermal matrix of sterile collagen origin, namely an Integra matrix (registered trademark) rinsed beforehand with Hank's Balanced Salt Solution (HBSS) three times, in a proportion of 30 000 fibroblasts per cm.sup.2 in a made-to-measure stainless steel incubation chamber. [0259] b. After 24 hours of culture at 37 C., 5% CO.sub.2, the incubation chamber was removed from the matrix. [0260] c. The seeded matrix was incubated at 37 C., 5% CO.sub.2 in DMEM including 10% of irradiated FCS and penicillin and streptomycin at 1 and 50 mg/mL ascorbic acid, for one week with medium changed every three days. [0261] d. Trypsinization of the keratinocytes and of the melanocytes with 0.025% trypsin and 0.0.1M EDTA for 1 to 2 minutes in order to detach the melanocytes from the melanocyte culture dishes, then for 10 minutes in order to detach the keratinocytes from the keratinocyte culture dishes. The melanocytes became detached first, which makes it possible to purify the cultures. Neutralization with irradiated FCS and centrifugation and seeding at 400 000 cells per cm.sup.2 in an incubation chamber of a mixture containing 1 melanocyte per 19 keratinocytes. [0262] e. Adhesion for 24 hours. [0263] f. Submersion for seven days in modified green medium: DMEM/Ham's F12/10% FCS including hyaluronic acid at 50 mg/ml. [0264] g. Interface for 7 days in modified Green medium: DMEM/Ham's F12 including 10% FCS, hyaluronic acid at 50 mg/ml and 50 mg/ml ascorbic acid and antibiotics, namely 1% penicillin-streptomycin.

[0265] FIG. 1 represents a diagram of the steps for obtaining a skin substitute.

[0266] In the present example, two skin equivalents obtained had (keratinocytes+melanocytes)/fibroblasts ratios of 13.3 respectively. For the ratio of 13.3, during the steps of depositing the fibroblasts and the keratinocytes/melanocytes mixture, the amounts of cells seeded were respectively 15 000 for the fibroblasts, 10 000 for the melanocytes and 190 000 for the keratinocytes.

[0267] Once the substitute/equivalent had been obtained, it was fixed in 4% formol, embedded in paraffin, then a 4 m section was cut, and then hematoxylin-eosin staining was performed in order to label the various layers of the skin. Observation under an optical microscope and at a magnification of 40 was carried out. Since the microscope was coupled to a CCD camera (Nikon, software NIS element Br), photographs of the observations were taken. FIG. 2B represents an optical microscopy photograph of a skin substitute obtained according to the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 13.3. FIG. 2C represents an optical microscopy photograph of a skin substitute obtained according to the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 6.7 and FIG. 2A represents an optical microscopy photograph of a normal skin biopsy. FIGS. 4A and 4B also represent optical microscopy photographs of a skin equivalent obtained according to the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 13.3.

[0268] Moreover, immunohistochemical labeling of the equivalent obtained by the method in which the (keratinocytes+melanocytes)/fibroblasts ratio was 13.3 and of an in vivo skin was carried out according to the method described in Salducci, M., Andr, N., Gur, C., Martin, M., Fitoussi, R., Vi, K., and Cario-Andr, M. (2014). Factors secreted by irradiated aged fibroblasts induce solar lentigo in pigmented reconstructed epidermis. Pigment Cell Melanoma Res. 27, 502-504 [12] or Simon, D., Daubos, A., Pain, C., Fitoussi, R., Vi, K., Taieb, A., de Benetti, L., and Cario-Andr, M. (2013). Exposure to acute electromagnetic radiation of mobile phone exposure range alters transiently skin homeostasis of a model of pigmented reconstructed epidermis. Int. J. Cosmet. Sci. 35, 27-34 [13] in order to identify in the equivalent the presence of melanocytes, the production of the basal lamina including in particular collagen IV (FIG. 4D(2)), the proliferative capacity of the cells in the basal lamina (FIG. 4D(1)) and the presence of melanocytes (FIG. 4C). FIGS. 4E and 4F represent optical microscopy photographs of skin in vivo after immunohistochemical labeling of the melanocytes in the basal position (FIG. 6E, light areas), labeling of collagen IV (FIG. 4F(2)) and labeling of the p63 proliferation marker (FIG. 4F(1)). It is clearly apparent on FIGS. 4C and 4D that the skin equivalent according to some embodiments includes melanocytes, and a basal lamina as demonstrated by the presence of collagen IV at the level of which the cells present are highly proliferative as for the skin in vivo (FIGS. 4E and 4F).

[0269] As represented on these photographs, the skin substitute/equivalent obtained by the method has a structure identical to that of the skin in vivo.

LIST OF REFERENCES

[0270] 1. Pendaries V et al., siRNA-mediated allele-specific inhibition of mutant type VII collagen in dominant dystrophic epidermolysis bullosa. JID 2012, June; 132(6):1741-3. [0271] 2. Petek L M et al., Efficient KRT14 targeting and functional characterization of transplanted human keratinocytes for the treatment of epidermolysis bullosa simplex. Mol ther 2010, September; 18 (9):1624-32. [0272] 3. Kogut et al., Differentiation of human induced pluripotent stem cells into a keratinocyte lineage Methods Mol Biol 2014, 1195:1-12. [0273] 4. Ohta et al., Generation of human melanocytes from induced pluripotent stem cells Methods Mol Biol, 2013; 989:193-215. [0274] 5. Revilla et al., Current advances in the generation of human iPS cells: implications in cell-based regenerative medicine. J Tissue Eng Regen Med, 2015, Mar. 11. [0275] 6. Bell et al., 1979 [0276] 7. Boyce S T et al., Structure of a collagen-GAG dermal skin substitute optimized for cultured human epidermal keratinocytes, 1988 October; 22 (10):939-57. [0277] 8. Hafemann et al., Use of a collagen/elastin-membrane for the tissue engineering of dermis. Burns 1999, August; 25(5):373-84. [0278] 9. Wonhye Lee et al., Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication. Biomaterials, 2009, March; 30(8):1587-95 [0279] 10. Pe{umlaut over (n)}a, and al., J Oral and Maxillofacial Surgery, 70:10 10, 2012 [0280] 11. E. Dantzer, F. Braye Reconstructive surgery using an artificial dermis (Integra): results with 39 grafts. Br J Plast Surg, 54:8 8, 2001. [0281] 12. Salducci, M., Andr, N., Gur, C., Martin, M., Fitoussi, R., Vi, K., and Cario-Andr, M. (2014). Factors secreted by irradiated aged fibroblasts induce solar lentigo in pigmented reconstructed epidermis. Pigment Cell Melanoma Res. 27,502-504 [0282] 13. Simon, D., Daubos, A., Pain, C., Fitoussi, R., Vi, K., Taieb, A., de Benetti, L., and Cario-Andr, M. (2013). Exposure to acute electromagnetic radiation of mobile phone exposure range alters transiently skin homeostasis of a model of pigmented reconstructed epidermis. Int. J. Cosmet. Sci. 35, 27-34