Process for creating a skin substitute and the resulting skin substitute

A process for preparing a skin substitute comprising dermis substitute and epidermis substitute comprising the steps of:

• (a) preparing a dermis substitute by admixing
• i) contractile cells collected from unilayer cultures comprising a nutritive medium implanted with animal cutaneous tissue comprising dermis and epidermis and
• ii) a culture nutritive medium to which has been added native type I collagen as a component,
• said mixture forming a gel which contracts, thereby expelling nutritive medium and forming said dermis substitute;
• (b) preparing an epidermis substitute by employing said dermis substitute as a substrate by culturing said substrate with keratinocytes from an animal extracted sample and maintaining conditions promoting the proliferation of keratinocytes on the surface of said substrate,
• (c) promoting the growth of said culture of said substrate with said keratinocytes such that a skin substitute is produced by employing at least one nutritive medium maintained in contact with said keratinocytes for a period of time of approximately 5-6 days,
• said substrate being implanted with at least one hair follicle or a section of a hair follicle extracted from animal skin,
• said follicle or follicle section retaining its cellular sheath and being implanted in said substrate in such a way that its median longitudinal line is substantially perpendicular to the surface of said substrate,
• (d) at the expiration of the 5-6 day immersion period, replacing said nutritive medium with another nutritive medium, the level of said another nutritive medium being raised to a point within the thickness of said dermis substitute,
• said dermis substitute being placed on a support grid raised from the bottom of said receptacle in order to ensure that said another nutritive medium reaches the desired level, and adjusting the level of said another nutritive medium so that said support grid is covered without, however, covering the upper surface of the skin substitute in its formative phase, and
• (e) extracting said hair follicle or section of a hair follicle from said dermis substrate at the end of approximately 8-13 days.

A process for preparing a skin substitute composed of a dermis equivalent covered with an epidermis equivalent, said dermis equivalent being a matrix formed from a type I collagen gel containing fibroblasts arranged three-dimensionally in said matrix, wherein said epidermis equivalent contains:

• (a) a basal membrane equivalent formed from the deposit of a layer of laminine, fibronectin and type IV collagen,
• (b) cells of the basal layer in a palisade arrangement, attached to the membrane equivalent according to paragraph (a) by hemidesmosomes and showing polar distribution of the bullous pemphigoid antigen;
• (c) cells of the suprabasal layers which, beginning with the first suprabasal layer, contain basic keratin with a value of 67 kDa, and acidic keratin with a value of 56.6 kDa;
• (d) granular cells containing involucrine, transglutaminase, filaggrine, keratohyaline granules and membrane coating granules, and
• (e) flat and keratinized cells arranged near the free surface, giving, after extraction using dodecyl sulfate and 2-mercaptoethanol, corneous envelopes characteristic of
• said follicle or fellicle section retaining its cellular sheath and being implanted in said substrate in such a way that its median longitudinal line is substantially perpendicular to the surface of said substrate.

A skin substitute comprising dermis substitute and epidermis substitute produced from the process comprising the steps of:

• (a) preparing a dermis substitute by admixing
• i) contractile cells collected from unilayer cultures comprising a nutritive medium implanted with animal cutaneous tissue comprising dermis and epidermis and
• ii) a culture nutritive medium to which has been added native type I collagen as a component,
• said mixture forming a gel which contracts, thereby expelling nutritive medium and forming said dermis substitute;
• (b) preparing an epidermis substitute by employing said dermis substitute as a substrate by culturing said substrate with keratinocytes from an animal extracted sample and maintaining conditions promoting the proliferation of keratinocytes on the surface of said substrate,
• (c) promoting the growth of said culture of said substrate with said keratinocytes such that a skin substitute is produced by employing at least one nutritive medium maintained in contact with said keratinocytes,
• said substrate being implanted with at least one hair follicle or a section of a hair follicle extracted from animal skin, wherein said hair follicle or section of a hair follicle is obtained by cutting an extracted hair follicle perpendicularly to its median longitudinal line, in the area in which said hair follicle remains enclosed in the cellular sheath thereof so as to obtain a length of between approximately 0.5 and 5 mm,
• said follicle or follicle section retaining its cellular sheath and being implanted in said substrate in such a way that its median longitudinal line is substantially perpendicular to the surface of said substrate, and
• (d) extracting said hair follicle or section of a hair follicle from said dermis substitute at the end of approximately 8-13 days.

A skin substitute comprising dermis substitute and epidermis substitute produced from the process comprising the steps of:

• (a) preparing a dermis substitute by admixing
• i) contractile cells collected from unilayer cultures comprising a nutritive medium implanted with animal cutaneous tissue comprising dermis and epidermis and
• ii) a culture nutritive medium to which has been added native type I collagen as a component,
• said mixture forming a gel which contracts, thereby expelling nutritive medium and forming said dermis substitute;
• (b) preparing an epidermis substitute by employing said dermis substitute as a substrate by culturing said substrate with keratinocytes from an animal extracted sample and maintaining conditions promoting the proliferation of keratinocytes on the surface of said substrate,
• (c) promoting the growth of said culture of said substrate with said keratinocytes such that a skin substitute is produced by employing at least one nutritive medium maintained in contact with said keratinocytes for a period of time of approximately 5-6 days,
• said substrate being implanted with at least one hair follicle or a section of a hair follicle extracted from animal skin,
• said follicle or follicle section retaining its cellular sheath and being implanted in said substrate in such a way that its median longitudinal line is substantially perpendicular to the surface of said substrate,
• (d) at the expiration of the 5-6 day immersion period, replacing said nutritive medium with another nutritive medium, the level of said other nutritive medium being raised to a point within the thickness of said dermis substitute,
• said dermis substitute being placed on a support grid raised from the bottom of said receptacle in order to ensure that said another nutritive medium reaches the desired level, and adjusting the level of said another nutritive medium so that said support grid is covered without, however, covering the upper surface of the skin substitute in its formative phase, and
• (e) extracting said hair follicle or section of a hair follicle from said dermis substrate at the end of approximately 8-13 days.