MX2014001357A - Process for generating a functional dermal substitute with pigmentation and hair follicles and use thereof as a regenerative agent. - Google Patents

Abstract

The present invention refers to a process for generating a functional dermal substitute with pigmentation and hair follicles and use thereof as a regenerative agent, which is obtained from the culture in vitro of human skin cells to be combined with gelling biodegradable and bio-compatible matrixes applied to diabetic foot, burns and skin cancer. The process comprises the following stages: obtaining a silicon resin solution, incubating the silicon resin solution, separating the dermis and epidermis, obtaining melanocytes, incubating the epidermis layer, disintegrating the dermis layer, disintegrating the epidermis layer, proliferating the keratinocytes in suspension, obtaining follicle dermal cells, separating the sterile biomass, generating cellular aggregates, preparing the functional dermal substitute and inoculating and proliferating the keratinocytes and melanocytes in the primary dermal substitute.

Description

PROCESS TO GENERATE A FUNCTIONAL DERMIC SUBSTITUTE WITH PIGMENTATION AND PILLOW FOLLICLES AND ITS USE AS AN AGENT REGENERATIVE.

DESCRIPTION OBJECT OF THE INVENTION The object of the invention lies in the process to generate a functional dermal substitute with picmentation and hair follicles from the in vitro culture of human skin cells to be combined with biodegradable and biocompatible gelling matrices and their use as a regenerative agent, preferably of the diabetic foot, burns and skin cancer.

BACKGROUND Currently, the use of allogeneic grafts and xenografts limits the regeneration of the skin due to inflammatory processes that originate from the immunological rejection that involves the use of donor skin and the use of skin from animals. This causes the low availability of said biological material for the treatment of skin wounds and there is the disadvantage of the susceptibility to infections. Currently in the market there are dermal substitutes based on the use of Silicon pads that help regenerate and fade skin scars caused by trauma and surgical procedures (Cicatricure Silicon Plates). So too, we can find the same product on the market called ScarAway and its use is based on the same principle as the aforementioned pads based on silicone. This techology of silicone pads are based on the constant pressure of the wound or scar for the stimulation of the regeneration of the skin. However, there is another variety of products based on the use of these pads with an added value generating a bi-layer based on a nylon network of bovine collagen (Biobrane &Integra) that will be in contact with the skin for the stimulation of the regeneration protected by said silicone pads mentioned above in the above products. (ScarWay &Cicatricure silicone patches).

However, we can find in the market other products based on the in vitro culture of human keratinocytes on latices of bovine collagen type 1 previously seeded with human fibroblasts (Apligraf).

Another approach to the capitalization and regeneration of the wound that has been developed in the laboratory is based on the in vitro culture of human keratinocytes on layers of fibroblasts from mouse embryos with a high degree of proliferation. (Epifast) BRIEF DESCRIPTION OF THE FIGURES Figures 1: General scheme of the composition of the functional dermal substitute. Figures 2: Gel layer of the cutaneous solution where fibroblasts and hair follicles are located.

Figure 3: Histological section and hematoxylin and eosin staining of a human skin sample as a positive control.

Figure 4: Histological section and hematoxylin and eosin staining of the functional dermal substitute after being cultured for 4 weeks.

Figure 5: Immunohistochemistry for the MEL-5 protein on a histological section of a human skin sample as a positive control.

Figure 6: Immunohistochemistry for the MEL-5 protein on a histological section of the functional dermal substitute.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a process for generating a functional dermal substitute with pigmentation and hair follicles (figure 1, 2, 3, 4 and 5) and its use as a regenerative agent in dermal pathologies, capable of producing re-pigmentation in lesions cutaneous, hair follicles to generate new hair, which is comprised of the following stages: a) Obtaining a silicone resin solution.

Use the Sylgard 184 elastomer solution pack protocol in sterile conditions to obtain a silicone resin solution. b) Incubation of the silicone resin solution.

Deposit 50% of the silicone resin solution within a volume in a cell culture container, preferably 115 cm2, to obtain a layer silicone of 10.72 cm x 10.72 cm and a thickness of 1000 mm and stand at a temperature of 37 ° C for 24 hours to obtain a silicone film.

After 24 hours at 37 ° C, remove the silicone film with tweezers carefully and sterilize the silicone film in a steam autoclave at a temperature of 120 ° C for 30 minutes. c) Separation of the dermis and epidermis.

Obtain a patient's skin sample in sterile conditions by means of a dermatome to obtain a sheet of skin, preferably 0.015 inches. Subsequently, wash the skin sample at least 3 times with a saline solution of PBS 1X phosphates containing 10% v / v of an antibiotic / antifungal solution. Then wash at least 3 times with a 1X PBS solution containing 1% v / v antibiotic / antifungal. Preferably, the antibiotic / antifungal solution is streptomycin / penicillin. After washing the skin sample, incubate with a cell disintegration proteolytic enzyme, preferably dispase, at a concentration of 1.79 units / mg under temperature conditions of 4 ° C for at least 16 hours to obtain a layer of epidermis and a Dermis layer. After 16 hours, separate the two layers by the use of sterile forceps and place them in sterile, independent containers. d) Obtaining melanocytes.

Scrape with a scalpel the basement membrane that is located in the lower part of the epidermis layer in the same container where the epidermis layer is. Subsequently, add 6% of a differentiation medium for melanocytes on the volume of the container to obtain monolayer melanocytes. e) Incubation of the epidermis layer.

Separating and washing the epidermal layer in a separate container at least 3 times with solution 1X PBS and incubating said layer of epidermis in a trypsin solution at a concentration of 0.025% trypsin in a final volume of ethylenediaminetetraacetic acid (EDTA) by 20 minutes at a temperature of 37 ° C to obtain a trypsinized epidermis. f) Disintegration of the dermis layer Place the dermis layer in a container and mechanically disintegrate with a sharp pulse tool in sterile conditions to obtain pieces of dermis of a maximum of 1mm x 1mm. After the incubation time of at least 10 minutes, carefully add 33.33% of the volume of the container of a cell culture medium for fibroblasts by the walls of the container and without moving the fragments of the dermis and incubate at 37 ° C, 95% Relative humidity, 5% CO2 and 20% 02 to obtain fibroblasts. g) Disintegration of the epidermis layer.

Neutralize the trypsinized epidermis with 50% of a culture medium of human keratinocytes containing 10% fetal bovine serum and shake vigorously for at least 30 seconds to obtain a cellular solution. Subsequently, filter the cell solution by a nitrocellulose filter with a pore size of 40pm to obtain the human keratinocytes in suspension in the filtrate. h) Proliferation of keratinocytes in suspension.

Seed the keratinocytes in a container with a cell culture medium for keratinocytes to obtain a greater quantity of keratinocytes in suspension. i) Obtaining follicular dermal cells.

Obtain a scalp biopsy of maximum 1 x 1 cm under aseptic conditions and place it in a container. Subsequently, wash the scalp biopsy at least 3 times with a 1x PBS solution containing 10% v / v of an antibiotic / antifungal solution, preferably streptomycin / penicillin. Then, wash another 3 times with a 1x PBS solution containing 5% (v / v) of the antibiotic / antifungal solution, preferably streptomycin / penicillin to obtain a sterile biopsy. j) Separation of the sterile biopsy.

Under stereoscopic illumination, place the container and mechanically and carefully separate the hair follicles from the sterile biopsy with the use of forceps and cutting pulse tool in sterile conditions to obtain the hair follicles. Subsequently and carefully, compress the hair follicles using the cutting pulse tool and cut a leaf from the capsule of the connective tissue sheath of the hair follicle and 10 minutes, then add cell culture medium for follicular dermal cells and then incubate at 37 ° C, 95 ° C. % relative humidity and 5% CO2 in sterile conditions to obtain follicular dermal cells.

The incubation of the melanocytes, keratinocytes, fibroblasts and follicular dermal cells is carried out for a period of 2-4 weeks in aseptic conditions with changes of fresh cell culture medium every second or third day until reaching a 100% cell confluence. k) Generation of cellular aggregates.

Deposit at least 1,200,000 follicular dermal cells in a well of an aggrewell box and centrifuge at 400 g x 3 minutes to obtain 1000 cell aggregates in each well. Subsequently, incubate the cell aggregates at a temperature of 37 ° C, 5% CO2 and 95% relative humidity for at least 24 hours. Then, obtain the cell aggregates from the wells by pipetting up and down 2 or 3 times with a 1X PBS solution and filter the contents of pipette through a nitrocellulose filter with pore size 37pm to remove the dissolved cells. Flip the nitrocellulose filter and deposit the cell aggregates present in said nitrocellulose filter in a sterile tube under aseptic conditions to obtain cell aggregates cell-free without adhesion. Then deposit in a Petri dish with low or no cell adhesion cell aggregates free of cells without aion to keep them for 4 days with culture medium for dermal follicle cells.

I) Preparation of the functional dermal substitute Deposit the previously sterilized silicone film in a container under aseptic conditions. Subsequently, deposit on the silicone film 56 ml of a skin solution for each 115.77 cm2 of silicone film. Said skin solution is composed of 27% v / v of a secondary solution composed of 225 grams / ml of bovine blood fibrinogen dissolved in phosphate buffered saline at a concentration of 1x, 27% v / v ml of type I collagen. a concentration of 3.1 mg / ml dissolved in a phosphate buffer saline at a concentration of 1x adjusting to pH 7.2 in aseptic conditions, 1 x 106 fibroblasts obtained in stage f), 6000 cell aggregates obtained in stage k), 1.8 % v / v mi of 1% CaCl2, 0.18% v / v of trenaxemic acid in a concentration of 1 mg / ml and 45% v / v mi of cell culture medium for fibroblasts, and 27% v / v of trompina with a concentration of 11 U / ml, on a final volume under sterile conditions, and then the container is shaken until the solidification of the cutaneous solution (figure 2) on the silicone film g to obtain a primary dermal substitute. Incubate the primary dermal substitute for a maximum period of 2 hours at 37 ° C at 95% relative humidity and 5% CO2. Subsequently, add 25% of the container volume of a cell culture medium for keratinocytes and incubate at a temperature of 37 ° C to 95% relative humidity and 5% C02, for at least 12 hours to obtain an enriched dermal substitute. m) Inoculation and proliferation of keratinocytes and melanocytes in the primary dermal substitute.

Aspirate the cell culture medium for keratinocytes and rest under aseptic conditions the enriched dermal substitute for at least 10 minutes, and then inoculate 1 x 106 melanocytes and 1 x 106 keratinocytes obtained in step d) and h) on the surface of the dermal substitute enriched and add 12.5% on the volume of the container of a cellular medium for keratinocytes and melanocytes. Then, incubate at 37 ° C, 5% C02, 90% relative humidity for at least 1 week to obtain a functional dermal substitute with pigmentation and hair follicles (figure 1, 3, 4, 5 and 6).

EXAMPLE 1. PREFERENTIAL MODE OF THE INVENTION.

The functional skin substitute with pigmentation and hair follicles is composed of a skin solution that is prepared by combining 225 grams of bovine blood fibrinogen in 15 ml (v / v) phosphate buffered saline at a concentration of 1x to obtain a 15mg solution / ml (w / v), 1 x 106 human fibroblasts, 6000 cellular aggregates of the follicular dermal cells, 1ml of 1% Ca (w / v), 10OmI of trenaxemic acid at a concentration of 1mg / ml (v / v) and 25 ml (v / v) of cell culture medium for fibroblasts to obtain a total solution of 56.1 mi. Subsequently, the solution is deposited on the previously sterilized silicone film and placed in a 115cm2 cell culture bottle. The gelling of the dermal scaffold based on bovine fibrinogen will be carried out by the addition of a 15 ml (v / v) thrombin solution of bovine lung or autologous human plasma at a concentration of 11 U / ml (v / v) by gently shaking the cell culture flask to generate a uniform coagulation of the solution and result in gelation of the cutaneous solution (FIG. ) on the silicone film. The total volume used for the gelation will be 56.1 ml obtaining a thickness of 5mm of the dermal scaffold.

EXAMPLE 1. MODE OF HANDLING THE FUNCTIONAL DERMAL SUBSTITUTE Under aseptic conditions, the cell culture bottle placed horizontally is opened and through the use of tweezers, the silicone film containing on its surface the gelled skin solution (embedded fibroblasts and follicular dermal aggregates) in fibrinogen is detached gently and at the 4 corners. / collagen or acellular plasma) and the epidermal layer (keratinocytes and melanocytes) previously cultured for 2 weeks. Once the dermal substitute has been obtained, it can be transported in sterile and sealed petri dishes to the operating room to be implanted in patients with cutaneous pathologies.

EXAMPLE 2. PREFERENTIAL USE OF THE INVENTION.

This functional dermal substitute is based on the well-orchestrated combination of the 4 cell types (fibroblasts, keratinocytes, melanocytes and follicular dermal cells) that make up the native and functional skin through the production of type I and IV collagen fibers (fibroblasts), production of melanin or cutaneous pigmentation (melanocytes), production of keratin (keratinocytes) and the generation of new hair follicles (cellular aggregates of dermal follicle cells). All this, in combination with the use of synthetic materials (silicone) that will provide flexibility, support and manageability to the dermal (two-dimensional) substitute to be implanted. Adding to this, also the novel use of biocompatible biomaterials (Fibrinogen from human blood, bovine blood fibrinogen and human collagen type I) with the human body that will stimulate cell proliferation in the dermal substitute, will generate vascularization at the time of implantation since it has growth factors and will stimulate the production of new extracellular matrix in the wounds treated in patients with cutaneous pathologies, increasing the re-epithelialization and skin regeneration time.

The dermal substitute replaces the use of alo, xeno and autografts since many times obtaining the skin of the same patient or the use of skin from banks is limited or not available without taking into account the use of skin of other species ( bovine origin).

The preferential use of this innovation is based on the implantation of the dermal substitute in cutaneous lesions with the potentiality of creating new cutaneous extracellular matrix together with its functionality (pigmentation and hair follicles.) Its mechanism of action is based on a skin lesion caused by the removal of cancerous tissue, first, second and third degree skin burn, the presence of a diabetic ulcer where the result is damaged, open skin susceptible to infections.

The dermal substitute is based on re-epithelialization and regeneration of skin damage.

This same (dermal substitute) is incorporated and / or fused with the skin damage by contact of the dermal layer (fibrin network with fibroblasts and follicular dermal cell aggregates) with the wound. From this first contact, the skin substitute dermal substitute fills the damage created on the skin regardless of the exposed thickness by providing on the surface of the lesion a coating of the silicone film that will provide protection against the environment and prevent the loss of water from the lesion The gelled cutaneous solution that is in contact with the cutaneous lesion, will provide over time a re-epithelialization and a regeneration sooner sooner by the addition of the live fibroblasts embedded within this fibrin network ( human fibrinogen, human acellular plasma, bovine fibrinogen / collagen type I) stimulating new generation of extracellular matrix and fibers of Collagen. In addition to this, the incorporation of the cellular aggregates of the follicular dermal cells within this fibrin network, will provide an early regeneration of the hair follicles for the generation of new hair in the opening lesion.

Subsequently, the intermediate layer between the silicone film and the cutaneous layer that is in contact with the lesion, will provide an early re-epithelialization thanks to the presence of human keratinocytes. In addition to this, the basal presence of the melanocytes will provide a re-pigmentation of the skin damage or injury.

EXAMPLE 3. ALTERNATIVES OF THE PROCESS Optionally, the isolation of the skin cells can also be obtained by means of a skin biposia by a 3 mm pulse and / or through a foreskin donated as a newborn. Also, all cell lines (fibroblasts, keratinocytes, melanocytes and follicles dermal) can be purchased from a bank of preserved cryo donor cell lines.

Optionally, the cellular aggregates are obtained by the hanging-drop method, a cellular pellet of the follicular dermal cells is obtained after 4 weeks of expansion, by washing 1 × PBS from the cell culture vial, trypsinization with trypsin 0.05%. EDTA for 5 minutes at 37 ° C, neutralizing the digestion with cell culture medium for follicular dermal cells with 10% (v / v) of fetal bovine serum and centrifuging the whole solution 1500 rpm for 5 minutes. Afterwards, the entire medium is sucked up so that only a cell pellet remains. After obtaining 30mI of the cell pellet, spheres or drops of the follicular dermal cells are placed inside a 100cm2 Petri dish lid of no cell culture in open or inverted position. Subsequently, PBS 1x (w / v) is added to the Petri dish and the lid is again placed in its original position so that 3 minutes later the drops fall (in the form of cell aggregates) in the 1x PBS suspension.

Optionally, the dermal scaffold can be generated by a cryopreserved human platelet serum (plasma acellular serum) from a blood sample centrifuged at 400 g for 5 minutes and resuspending 15 ml of the plasma acellular serum with 1 x 106 human fibroblasts, 6000 Cellular aggregates of follicular dermal cells 1ml of 1% CaCl2 (w / v), 100mI of trenaxemic acid at a concentration of 1mg / ml (v / v) and 25 ml (v / v) of cell culture medium for fibroblasts to obtain a total solution of 40.1 mi. The solution is then deposited on the previously sterilized silicone resin film and placed in a 115cm2 cell culture flask. The gelation of the dermal scaffold based on plasma acellular serum (cryopreserved platelet) will be carried out by the addition of a 15 ml (v / v) solution of bovine lung thrombin or thrombin from autologous human plasma at a concentration of 11 U / ml (v / v) by gently shaking the cell culture bottle to generate a uniform coagulation of the solution and to obtain as a result the gelling of the cutaneous solution on the film of silicone. The total volume used for the gelation will be 56.1 ml (v / v) obtaining a thickness of 5mm of the dermal scaffold.

After gelation in 5 minutes, the cell culture bottle is incubated at 37 ° C, 5% CO2, 90% relative humidity for 2 hours. Subsequently 10 ml (v / v) of cell culture medium for human keratinocytes is added and incubated at 37 ° C, 5% C02, 90% relative humidity for 24 hours.

Claims (19)

CLAIMS Having described my invention enough, I consider it as a novelty and therefore claim as my exclusive property, what is contained in the following clauses:

1. A process for generating a functional dermal substitute with pigmentation and hair follicles characterized because it is rised of the following stages: a) Obtaining a solution of siiicone resin, b) incubation of the siiicone resin solution, c) separation of the dermis and the epidermis, d) obtaining melanocytes, e) incubation of the epidermis layer, f) disintegration of the dermis layer, g) disintegration of the epidermis layer, h) proliferation of keratinocytes in suspension, i) obtaining follicular dermal cells, j) separation of the sterile biopsy, k) generation of cellular aggregates, l) preparation of the functional dermal substitute, and m) inoculation and proliferation of the keratinocytes and melanocytes in the primary dermal substitute.

2. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step a), the Sylgard 184 elastomer solution pack protocol is used under sterile conditions to obtain a resin solution of sylicon.

3. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step b), 50% of the silicone resin solution is deposited within a volume in a cell culture container, preferably of 115 cm2, to obtain a silicone layer of 10.72 cm x 10.72 cm and a thickness of 1000 mm and it rests at a temperature of 37 ° C for 24 hours to obtain a silicone film; after 24 hours at 37 ° C, the silicone film is carefully removed with tweezers and the silicone film is sterilized in a steam autoclave at a temperature of 120 ° C for 30 minutes.

4. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step c), a patient's skin sample is obtained in sterile conditions by means of a dermatome to obtain a skin lamina , preferably 0.015 inches; Subsequently wash the skin sample at least 3 times with a phosphate solution PBS 1X containing 10% v / v of an antibiotic / antifungal solution; then, it is washed at least 3 times with a 1X PBS solution containing 1% v / v of antibiotic / antifungal; after washing the skin sample, it is incubated with a cellular disintegration proteolytic enzyme, at a concentration of 1.79 units / mg under temperature conditions of 4 ° C for at least 16 hours to obtain a layer of epidermis and a layer of dermis; after 16 hours, the two layers are separated by the use of sterile forceps and placed in sterile, independent containers.

The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 4, characterized in that the antibiotic / antifungal solution is preferably streptomycin / penicillin.

6. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 4, characterized in that the cellular disintegration proteolytic enzyme is preferably disparate.

7. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step d), the basal membrane located in the lower part of the epidermis layer in the skin is scraped with a scalpel. same container where the epidermis layer is; subsequently, 6% of a differentiation medium for melanocytes is added to the volume of the container to obtain monolayer melanocytes.

8. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step e), the epidermis layer is separated and washed in a separate container at least 3 times with the PBS 1X solution and said epidermis layer is incubated in a trypsin solution at a 0.025% concentration of trypsin in a final volume of ethylenediaminetetraacetic acid (EDTA) for 20 minutes at a temperature of 37 ° C to obtain a trypsinized epidermis.

9. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step f), the dermis layer is placed in a container and mechanically disintegrated with a cutting pulse tool under sterile conditions for get pieces of dermis like maximum 1mm x 1mm; After the incubation time of at least 10 minutes, 33.33% of the container volume of a cell culture medium for fibroblasts is carefully added through the walls of the container and without moving the fragments of the dermis and incubate at 37 ° C, 95% Relative humidity, 5% CC > 2 and 20% Ü2 to obtain fibroblasts.

10. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step g), the trypsinized epidermis is neutralized with 50% of a culture medium of human keratinocytes containing fetal bovine serum. % and stir vigorously for at least 30 seconds to obtain a cellular solution; subsequently, the cell solution is filtered through a nitrocellulose filter with a pore size of 40pm to obtain the human keratinocytes in suspension in the filtrate.

11. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step h), the keratinocytes are seeded in a container with a cell culture medium for keratinocytes to obtain a greater quantity of keratinocytes in suspension.

12. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step i), a scalp biopsy of maximum 1 x 1 cm is obtained under aseptic conditions and placed in a container; subsequently, the scalp biopsy is washed at least 3 times with a 1x PBS solution containing 10% v / v of an antibiotic / antifungal solution; then wash another 3 times with a 1x PBS solution containing 5% (v / v) of the antibiotic / antifungal solution to obtain a sterile biopsy.

13. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 12, characterized in that the antibiotic / antifungal solution is preferably streptomycin / penicillin.

14. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step j), the container is placed under stereoscopic illumination and the hair follicles are separated mechanically and carefully from the sterile biopsy with the use of forceps and cutting pulse tool in sterile conditions to obtain the hair follicles; subsequently and carefully, the hair follicles are compressed by the cutting pulse tool and a leaf is cut from the capsule of the connective tissue sheath of the hair follicle and 10 minutes, then adds cell culture medium for follicular dermal cells and then incubates at 37 ° C, 95% relative humidity and 5% CO2 under sterile conditions to obtain follicular dermal cells.

15. The process to generate a functional dermal substitute with pigmentation and hair follicles, characterized in that the incubation of melanocytes, keratinocytes, fibroblasts and follicular dermal cells is performed for a period of 2-4 weeks under aseptic conditions with changes of fresh cell culture medium every second or third day until a 100% cell confluence arrives.

16. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step k), at least 1,200,000 follicular dermal cells are deposited in a well of an aggrewell box and centrifuged at 400 gx 3 minutes to obtain 1000 cellular aggregates in each well; subsequently, the cell aggregates are incubated at a temperature of 37 ° C, 5% C02 and 95% relative humidity for at least 24 hours; then, the cell aggregates of the wells are obtained by pipetting up and down 2 or 3 times with a 1X PBS solution and filtering the contents of the pipette through a nitrocellulose filter with a pore size of 37pm to remove the dissolved cells; the filter is flipped of nitrocellulose and the cell aggregates present in said nitrocellulose filter are deposited in a sterile tube under aseptic conditions to obtain cellular aggregates free of cells without adhesion; Then the cellular aggregates free of cells without adhesion are deposited in a Petri dish of low or no cellular adhesion to maintain them for 4 days with culture medium for dermal follicle cells.

17. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in stage I), the previously sterilized silicone film is deposited in a container under aseptic conditions; subsequently, 56 ml of a skin solution is deposited on the silicone film for each 115.77 cm 2 of silicone film; said skin solution is composed of 27% v / v of a secondary solution composed of 225 grams / ml of bovine blood fibrinogen dissolved in phosphate buffered saline at a concentration of 1x, 27% v / v ml of collagen type I a concentration of 3.1 mg / ml dissolved in a phosphate buffer saline at a concentration of 1x adjusting to pH 7.2 in aseptic conditions, 1 x 106 fibroblasts obtained in stage f), 6000 cell aggregates obtained in stage k), 1.8 % v / v mi of CaCl2 at 1%, 0. 18% v / v of trenaxemic acid in a concentration of 1 mg / ml and 45% v / v mi of cell culture medium for fibroblasts, and 27% v / v of trompine with a concentration of 11 U / ml, on a final volume in conditions sterile, and subsequently the container is shaken until the solidification of the skin solution on the silicone film to obtain a primary dermal substitute; the primary dermal substitute is incubated for a maximum period of 2 hours at 37 ° C at 95% relative humidity and 5% CO2; subsequently, 25% of the container volume of a cell culture medium for keratinocytes is added and incubated at a temperature of 37 ° C at 95% relative humidity and 5% CO2, for at least 12 hours to obtain a dermal substitute enriched.

18. The process for generating a functional dermal substitute with pigmentation and hair follicles according to claim 1, characterized in that in step m), the cell culture medium for keratinocytes is aspirated and the enriched dermal substitute is rested under aseptic conditions for at least 10 minutes, and subsequently, 1 x 106 melanocytes and 1 x 106 keratinocytes obtained in step d) and h) are inoculated on the surface of the enriched dermal substitute and add 12.5% on the volume of the cell medium container for keratinocytes and melanocytes; then, incubate at 37 ° C, 5% C02, 90% relative humidity for at least 1 week to obtain a functional dermal substitute with pigmentation and hair follicles.

19. Use of the functional dermal substitute with pigmentation and hair follicles according to claims 1 to 18 as a regenerative agent.