[0001] The present invention relates to a method for the in vitro production of an organotypic skin model, wherein said skin model is able to complete skin maturation due to suitable culture media. This methodology provides with a new product that better resembles human skin, that can be cultured for long period of times, and that is suitable for in vitro testing and modelling.
1. A method for the in vitro production of an organotypic skin model, the method comprising the steps of: a) providing a solution comprising stromal cells, blood plasma, fibrinogen, and a source of calcium; and incubating said solution until a hydrogel is formed, b) adding to the hydrogel of step a), preferably on its surface, a cell population comprising keratinocytes, and culturing the hydrogel in a keratinocyte culture media to allow the keratinocytes to grow until at least 90% of confluency is reached, wherein the keratinocyte culture media is characterized by having a percentage of serum of more than 8% (v/v), c) transferring the product resulting from step b) to an air-liquid interface and culturing said product in a epidermal differentiation culture media to provide an organotypic skin model with epidermal differentiation, wherein the epidermal differentiation culture media is characterized by having a percentage of serum of less than 1% (v/v), and d) maturing the organotypic skin model with epidermal differentiation of step c) in an epidermal maintenance culture media, wherein the epidermal maintenance culture media is characterized by having a percentage of serum of between 4-6% (v/v). 2. The method according claim 1, wherein the step d) is carried out until the keratinocytes are differentiated as indicated by the expression in the epidermal layer of Keratin 5, Keratin 14, Keratin 10, Collagen IV, Laminin 5, Loricrin, Filaggrin and/or Involucrin proteins. 3. The method according to claim 1 or 2, wherein the resulting matured organotypic skin model of step d) is characterized by comprising: - an epidermal compartment, wherein the epidermal compartment is in the upper part of the organotypic skin model, and it comprises keratinocytes and hyaluronic acid, - a basement membrane, wherein said basement membrane is located below the epidermal compartment, and where it is characterized by being a membrane comprising collagen IV and laminin 5, - a dermal compartment, wherein the dermal compartment is located in the base of the organotypic skin culture, and it comprises fibroblasts, collagen III and I. 4. The method according to claim 3, wherein the epidermal compartment is characterized by comprising, from top to bottom: - a cornified epidermal layer or stratum corneum comprising cells, preferably corneocytes, and filaggrin, - a granular epidermal layer comprising cells, preferably keratinocytes, expressing loricrin, - a suprabasal (or spinous) epidermal layer comprising cells, preferably keratinocytes, expressing keratin 10 and involucrin, and - a basal epidermal layer comprising cells, preferably keratinocytes, expressing keratin 5 and 14. 5. The method according to any of claims 1 to 4, wherein the cell population comprising fibroblasts cells added in step a) are dermal fibroblasts from human foreskin. 6. The method according to any of claims 1 to 5, wherein the cell population comprising keratinocytes cells added in step b) are epidermal keratinocytes from human foreskin. 7. The method according to any of claims 1 to 6, wherein the final concentration of fibrin in the resulting hydrogel of a) is between 1.2-4.5 mg/mL, preferably 3.5 mg/mL. 8. The method according to any of claims 1 to 7, wherein the solution of plasma has a concentration of platelets of less than 10 × 10<5> platelets/µL. 9. The method according to any of claims 1 to 8, wherein an antifibrinolytic agent is further added to the solution of step a). 10. The method according to any of claims 1 to 9, wherein the source of calcium added in step a) is at a final concentration of between 0.01-2%, preferably 0.08% (w/v). 11. The method according to any of claims 1 to 10, wherein the total number of fibroblasts cells added in step a) is 5.000-80.000 cells/ml, preferably 20.000 cells/ml. 12. The method according to any of claims 1 to 11, wherein the total number of keratinocytes cells added in step b) is 12.000-1.500.000 cells/ml, preferably 1 × 10<6> cells/ml. 13. An organotypic skin model obtained or obtainable from the method according to claims 1 to 12. 14. In vitro use of an organotypic skin model obtained or obtainable from the method according to claims 1 to 13 in pharmacological development assays, chemical or pollutants toxicity assays, screening of medical and/or cosmetic drugs, testing mechanical irritations through textiles, basic study of the fundamental biology of skin, and/or the processes that mediate skin ageing or skin disease.