PROCESS FOR OBTAINING A PRE-VASCULARIZED DERMAL-EPIDERMAL TISSUE

Abstract

The invention relates to a process for obtaining a skin substitute, comprising the following steps: a) mixing fibroblasts, endothelial cells and hydrogel of exclusively biological origin; b) incubating the mixture obtained in step a) for a sufficient time and under suitable conditions to obtain a pre-vascularized dermis; c) adding keratinocytes to the pre-vascularized dermis of step b) to obtain a skin substitute; wherein said fibroblasts, endothelial cells and keratinocytes were obtained from pluripotent stem cells.

Claims

1. A process for obtaining a skin substitute, comprising the following steps: a) mixing fibroblasts, endothelial cells and hydrogel of exclusively biological origin; b) incubating the mixture obtained in step a) in a culture medium for a sufficient time and under suitable conditions to obtain a pre-vascularized dermis tissue; c) adding keratinocytes to the pre-vascularized dermis tissue of step b) to obtain a skin substitute; and d) optionally, incubating the skin substitute at an air-liquid interface for a sufficient time to obtain a pluristratified pre-vascularized dermal-epidermal tissue, wherein said fibroblasts, endothelial cells and keratinocytes were obtained from pluripotent stem cells.

2. The process of claim 1, wherein no vascular smooth muscle cell is added to the mixture of step a).

3. The process of claim 1, wherein the step of incubating takes place for 7 days.

4. The process of claim 1, wherein vascular epidermal growth factor (VEGF), preferably stabilized VEGF, is present in the culture medium of step b), preferably at a concentration from 30 to 60 ng/mL, preferably 45 to 55 ng/mL.

5. The process of claim 1, wherein VEGF, is present in the culture medium of step c).

6. The process of claim 1, wherein the fibroblasts and the endothelial cells of step a) are obtained from human induced pluripotent stem cells.

7. The process as of claim 1, wherein the keratinocytes of step c) are obtained from human induced pluripotent stem cells.

8. A skin substitute comprising a pre-vascularized dermis and a monolayer of keratinocytes obtained according to the process of claim 1.

9. A skin substitute comprising a pre-vascularized dermis and a pluristratified epidermis, obtained according to the process of of claim 1.

10. A method of treating a wound in a subject in need thereof, comprising grafting the skin substitute according to claim 8 onto the wound.

11. A method of treating a wound in a subject in need thereof, comprising grafting the skin substitute according to claim 9 onto the wound.

12. A method for screening for compounds and molecules intended to be in contact with the skin for cosmetic and/or therapeutic purposes, comprising the step of contacting the skin substitute as claimed in claim 8 with the compounds and/or molecules.

13. A population of skin substitutes obtained according to the process as claimed in claim 7.

14. A method for screening for compounds and/or molecules intended to be in contact with the skin and notably for cosmetic and/or therapeutic purposes, comprising the step of contacting the skin substitute as claimed in claim 9 with the compounds and/or molecules.

15. The process of claim 1, wherein stabilized vascular epidermal growth factor (VEGF), is present in the culture medium of step b).

16. The process of claim 1, wherein stabilized vascular epidermal growth factor (VEGF), is present in the culture medium of step b) at a concentration from 30 to 60 ng/mL.

17. The process of claim 1, wherein stabilized vascular epidermal growth factor (VEGF), is present in the culture medium of step b) at a concentration from 45 to 55 ng/mL.

18. The process of claim 1, wherein stabilized vascular epidermal growth factor (VEGF), is present in the culture medium of step c).

19. The process of claim 1, wherein stabilized vascular epidermal growth factor (VEGF), is present in the culture medium of step c) at a concentration from 30 to 60 ng/mL.

20. The process of claim 1, wherein stabilized vascular epidermal growth factor (VEGF), is present in the culture medium of step c) at a concentration from 45 to 55 ng/mL.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0045] Other characteristics, details and advantages of the invention will emerge from reading the appended figures.

[0046] FIG. 1 represents the process for obtaining dermis equivalent from endothelial cells, from fibroblasts and from fibrin.

[0047] FIG. 2 represents an example of the process for obtaining dermal-epidermal tissue according to the invention.

[0048] FIG. 3 represents an example of dermal-epidermal tissue according to the invention sutured onto a mouse.

[0049] FIG. 4 represents the formation of circular structures composed of endothelial cells in a dermal-epidermal tissue obtained according to the process of the invention in which the biomaterial is fibrin and the fibroblasts and endothelial cells derived from human iPSCs are seeded in a 1:40 ratio (EC:CNT medium).

EXAMPLES

[0050] Example 1: Obtaining of Fibroblasts, of Endothelial Cells and of Keratinocytes from Induced Pluripotent Stem Cells

[0051] A human line of induced pluripotent stem cells was used to obtain, via 3 suitable differentiation processes, fibroblasts (FIB), endothelial cells (EC) and keratinocytes (KER).

Example 2: Obtaining of a Pre-Vascularized Dermis

[0052] The fibroblasts (FIB) and the endothelial cells (EC): the FIB and the EC were preamplified upstream of the preparation of the tissues in culture flasks and in F-CnT medium (CelInTec) for the FIB and in StemPro34 (Invitrogen) or ENDO-CnT (CelInTec) medium supplemented with VEGF at 50ng/mL for the EC. When the 2 cell types were ready, they were passaged and counted. The cells at the ratio of 1 FIB for 40 EC were mixed with fibrin. The fibrin is obtained by mixing plasma and saline solution under the conditions described in Table 1.

TABLE-US-00001 TABLE 1 Fibrin components Initial concentrations Volume for 5 mL Human plasma —  2 mL NaCl 0.9%  2.6 mL Exacyl ® 0.1 g/mL  20 μL CaCl.sub.2 0.1M 400 μL

[0053] The “cells+fibrin” solution was deposited in inserts of 1 cm.sup.2, i.e. 600 μL/insert, with 10 000 FIB+400 000 EC. The plates were then placed at +37° C. to allow coagulation of the matrix for a minimum of 1 h. When the fibrins had set, medium was added: 1 mL in the inserts and 2 mL in the wells so that the tissue is completely immersed. The medium used is a ⅓ mixture of F-CnT medium and ⅔ of “EC” medium for 1 day. The following day (D1) and up to D7, the medium was changed every 2 days for the medium composed of ⅓ of F-CnT medium and ⅔ of EC medium, with the same volumes as mentioned.

Example 3: Obtaining of a Pre-Vascularized Dermal-Epidermal Tissue

[0054] The culture was carried out across approximately 14 days in an incubator regulated at +37° C. with 5% CO.sub.2 for the dermal tissues and 21 days for the dermal-epidermal tissues. The F-CnT medium of the mixture was replaced with the ECM medium on D1 to allow the FIB to secrete extracellular matrix and stabilized VEGF at 50 ng/mL.

[0055] On D7, a part of the tissues was left under the same conditions up to D14 in order to perform an analysis of the dermis alone, and the other part was seeded with keratinocytes (KER) on the matrices in CnT07.HC medium with 1.6 mg/mL of Exacyl® for 24-48 h at 100 000/cm.sup.2. On D9, the tissues with KER were placed at the air-liquid interface to allow stratification of the epidermis. The medium used was then composed of Airlift medium at 70%, ENDO-CnT medium at 30% with Exacyl® at 1.6 mg/mL and stabilized VEGF at 50 ng/mL.

[0056] These experiments made it possible to obtain a pre-vascularized dermal-epidermal tissue which, once grafted onto the mouse, did not exhibit any necrosis.