SKIN EQUIVALENT WITH DISTINCT JUXTAPOSED DERMAL COMPARTMENTS

Abstract

The invention relates to a dermis equivalent comprising longitudinally at least two distinct juxtaposed dermal compartments of different compositions, along with a skin equivalent comprising this dermis equivalent.

Claims

1. A dermis equivalent comprising longitudinally at least two distinct, juxtaposed dermal compartments of different compositions.

2. The dermis equivalent according to claim 1, wherein at least one of the at least two dermal compartments comprises collagen that has undergone oxidative modification and at least another of the at least two dermal compartments comprises collagen that has not undergone oxidative modification, or that has undergone another oxidative modification different from the first compartment.

3. The dermis equivalent according to claim 1, wherein at least one of the at least two dermal compartments comprises papillary fibroblasts and at least another of the at least two dermal compartments comprises reticular fibroblasts.

4. A skin equivalent displaying surface heterogeneity, comprising a dermis equivalent according to claim 1.

5. A process for the preparation of a dermis equivalent according to claim 1, comprising preparing at least two distinct, longitudinally juxtaposed lattices of different compositions, one of the at least two lattices being prepared inside a mold, while the other is prepared on the periphery of the mold.

6. The process according to claim 5, wherein the preparing of the at least two lattices comprises the preparation of at least two distinct solutions of different compositions, one being for the inside the mold and the other being for the periphery of the mold.

7. A process of preparation of a skin equivalent according to claim 4, comprising preparing at least two distinct, longitudinally juxtaposed lattices of different compositions, one of the at least two lattices being prepared inside a mold, while the other is prepared on the periphery of the mold.

8. A process for the preparation of a skin equivalent displaying surface heterogeneity which comprises using the dermis equivalent according to claim 1.

9. A process for the study of skin heterogeneity which comprises using the dermis equivalent according to claim 1, or of a skin equivalent displaying surface heterogeneity, comprising said dermis equivalent.

10. A process of screening for a compound displaying activity following topical application onto the skin comprising the application of a candidate compound onto the dermis equivalent according to claim 1, or onto a skin equivalent displaying surface heterogeneity, comprising said dermis equivalent.

11. The dermis equivalent according to claim 2, wherein at least one of the at least two dermal compartments comprises papillary fibroblasts and at least another of the at least two dermal compartments comprises reticular fibroblasts.

12. A skin equivalent displaying surface heterogeneity, comprising a dermis equivalent according to claim 11.

13. A skin equivalent displaying surface heterogeneity, comprising a dermis equivalent according to claim 2.

14. A skin equivalent displaying surface heterogeneity, comprising a dermis equivalent according to claim 3.

15. A process for the preparation of a dermis equivalent according to claim 2, comprising preparing at least two distinct, longitudinally juxtaposed lattices of different compositions, one of the at least two lattices being prepared inside a mold, while the other is prepared on the periphery of the mold.

16. A process for the preparation of a dermis equivalent according to claim 3, comprising preparing at least two distinct, longitudinally juxtaposed lattices of different compositions, one of the at least two lattices being prepared inside a mold, while the other is prepared on the periphery of the mold.

17. A process for the preparation of a dermis equivalent according to claim 11, comprising preparing at least two distinct, longitudinally juxtaposed lattices of different compositions, one of the at least two lattices being prepared inside a mold, while the other is prepared on the periphery of the mold.

18. The process according to claim 7, wherein the preparing of the at least two lattices comprises the preparation of at least two distinct solutions of different compositions, one being for the inside the mold and the other being for the periphery of the mold.

19. A process of screening for a compound displaying activity following topical application onto the skin comprising the application of a candidate compound onto the dermis equivalent according to claim 2 or onto a skin equivalent displaying surface heterogeneity, comprising said dermis equivalent.

20. A process of screening for a compound displaying activity following topical application onto the skin comprising the application of a candidate compound onto the dermis equivalent according to claim 3 or onto a skin equivalent displaying surface heterogeneity, comprising said dermis equivalent.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0080] FIG. 1: Schematic representation of the skin equivalent according to the invention, comprising the dermis equivalent, longitudinally comprising at least two distinct juxtaposed dermal compartments of different compositions according to the invention.

[0081] FIG. 2: (A) Histology of the skin equivalent obtained in the example, comprising 3 distinct juxtaposed dermal compartments: one compartment comprising reticular fibroblasts surrounded by two compartments comprising papillary fibroblasts. (B) Labeling of the filaggrin present in the skin equivalent obtained in the example.

EXAMPLE

[0082] This example shows a reconstructed skin displaying alternately, at the dermal level, zones with papillary or reticular fibroblasts.

Preparation of the Dermis Equivalent With Three Distinct Compartments

[0083] Two human fibroblasts solutions, one comprising papillary fibroblasts, the other comprising reticular fibroblasts, are prepared in MEM 25 mM Hepes medium with 10% FCS at a rate of 210.sup.6 cells/ml.

[0084] From these fibroblast solutions, two distinct lattice preparation solutions are prepared, one (solution A) for the periphery of a 1.5 cm3.8 cm rectangular Teflon inclusion mold, and the other (solution B) for the interior of this mold.

[0085] Solution A comprises 3.22 ml of MEM 1.76 medium, 0.63 ml FXS, 0.35 ml NaOH 0.1 N, 0.2 ml of MEM 25 mM Hepes medium, 10% FCS and 0.5 ml of papillary fibroblasts solution.

[0086] Solution B comprises 3.22 ml MEM 1.76 medium, 0.63 ml FXS, 0.35 ml NaOH 0.1 N and 0.2 ml of MEM 25 mM Hepes medium, 10% FCS and 0.5 ml reticular fibroblasts solution.

[0087] 2.1 ml of cold acid-soluble collagen I solution (5 mg/ml) is slowly added to solutions A and B.

[0088] The solutions are mixed with a pipette until homogenization.

[0089] The rectangular mold is placed at the center of a 4 cm4 cm square microscope dish covered with a solution comprising 3.22 ml of MEM 1.76 medium, 0.63 ml FXS, 0.35 ml NaOH 0.1 N and 0.2 ml of MEM 25 mM Hepes medium, 10% FCS. The assembly is incubated at 37 C.

[0090] 1.7 ml of solution B is poured into the mold and 5.3 ml of solution A is poured at the mold's periphery. The assembly is then incubated for 15 to 20 min, to allow gelling to occur.

[0091] Once gelling is obtained, the mold is removed and the dish transferred to 37 C., 5% CO.sub.2.

[0092] The preparation is then incubated at 37 C., 5% CO.sub.2 for 3 days to allow the lattices to contract.

Skin Equivalent Preparation

[0093] Seeding with keratinocytes to obtain the complete skin equivalent is performed after 3 days of lattice contraction.

[0094] An adhesive solution is prepared by mixing 0.46 ml of MEM 1.76 medium, 0.09 ml FCS, 0.05 ml NaOH 0.1N, 0.1 ml MEM Hepes 10% SVF medium and 0.3 ml dialyzed collagen.

[0095] A drop of this solution is deposited in the middle of a culture dish. The lattices comprising the 3 dermal compartments prepared above are taken and placed onto the drop of adhesive solution, then incubated in the oven at 37 C., 5% CO.sub.2 for 20-30 min.

[0096] The keratinocytes are put in solution in MEM 10% FCS+3F medium at a concentration of 110.sup.5 cells/ml.

[0097] A seeding ring is placed onto the bonded lattices and 0.5 ml of keratinocyte cell suspension is deposited into this ring. MEM 10% FCS+3F medium is added around the ring.

[0098] The assembly is then incubated at 37 C., 5% CO.sub.2 for 2 h.

[0099] The seeding ring is then removed and the dishes re-incubated at 37 C., 5% CO.sub.2 for 7 days, the culture medium being changed twice per week.

[0100] After 7 days of immersion culture, the skins are emerged: the adhesive around the skin to emerge is cut and the skin equivalent transferred to an emersion mesh in a dish comprising MEM 10% FCS+3F medium.

[0101] The dishes are incubated at 37 C., 5% CO.sub.2 for 7 days, the culture medium being changed twice per week.

Analysis of the Obtained Skin Equivalents

[0102] The obtained skin equivalent was studied by histology and labeled to detect filaggrin. As shown in FIG. 2, it is apparent in the histologies corresponding to the zones of dermal compartment with papillary fibroblasts and of dermal compartment with reticular fibroblasts, that the epidermal areas corresponding to these compartments can be distinguished by the quality of their differentiation, which is less pronounced for the reticular fibroblast compartment by comparison to the papillary fibroblast compartment.

[0103] This is confirmed, in the stratum granulosum, by the filaggrin labeling, which is stronger in the presence of a dermal part containing papillary fibroblasts, compared to a dermal part containing reticular fibroblasts.