FR2843972A1 - Skin substitute, useful for in vitro testing and for repairing skin wounds, comprises layer of skin cells adherent on a chitosan matrix - Google Patents

Abstract

A skin substitute (A) comprises a cellular layer (B), of keratinocytes and/or fibroblasts, adherent on a bioresorbable, three-dimensional matrix (C) based on chitosan, is new. Independent claims are also included for the following: (1) support (D) for cell cultures, for preparation of (A), in which the culture surface comprises (C) and has surface area at least 15 cm2; (2) cultures of adherent cells (keratinocytes, fibroblasts or stem cells) on (D); (3) methods for preparing (D); and (4) method for preparing (A) by culturing cells on (D).

Description

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BIOACTIVE SKIN SUBSTITUTES COMPRISING A SHEET OF
CELLS GROWN ON A MATRIX # BASED WITH CHITOSANE,
The present invention relates to skin substitutes consisting of a cell sheet obtained by culturing fibroblasts and / or human keratinocytes on a hydrated, flexible and bioresorbable matrix, based on chitosan, poured into the bottom of a culture vessel. The cell sheet, which is preferably multilayer, can consist of epithelial cells (equivalent epidermis), fibroblasts (equivalent dermis) or be a mixed sheet of epithelial cells and fibroblasts (reconstructed skin). The chitosan-based biomaterial covered with a multilayer skin cell sheet, can find therapeutic applications as a bioactive cell dressing for the treatment of chronic wounds (ulcers, bedsores), for the treatment of acute wounds (wounds, burns), as healing agent, pharmacological or even cosmetic applications, and applications in in vitro toxicology tests.

 The treatment of severe skin lesions such as second and third degree burns, ulcers and bedsores, has two main aspects: on the one hand, it is necessary to quickly apply a bandage to avoid dehydration and infections and, d On the other hand, it is necessary to stimulate the regeneration of the epidermis or more generally of the skin. Currently, in the case of very serious third degree burns on less than half of the body surface, an autograft of the patient's skin is performed after excision of the burned skin. In this case, healthy skin is removed from the patient, passed through a machine which makes it possible to increase the surface (principle of wicked skin) and grafted onto the patient at the level of the lesions. In the case of very serious third degree burns on more than half of the body surface, there is no longer enough healthy skin surface to obtain chewed skin and perform the autograft. We must first

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 temporarily cover wounds to prevent dehydration and infection, using corpse skin or skin substitutes. Skin cells of the patient are meanwhile cultured in vitro, to obtain multilayer epithelial sheets which are then grafted.

 The techniques currently used to cultivate epidermal leaflets for the grafting of burn victims, allow to obtain in three weeks from 1 to 2 m2 of epidermis, from a few square centimeters of skin (biopsy). The sheets of keratinocytes cultivated in vitro undergo an enzymatic treatment with the dispase to detach them from the bottom of the culture dish. These extremely fragile sheets must then be stapled onto a vaseline pad, before being grafted onto the patient's wounds.

 Acellular dressings, intended for the temporary covering of wounds of burn victims, or to cover small wounds while waiting for the natural regeneration of the skin, are the subject of intense research. Examples include dressings made from dextran or xanthan type polysaccharides in the form of a hydrated film, incorporating, if necessary, factors promoting healing (US Pat. No. 6,132,759). Recently, a flexible polymer film based on chitosan, intended to serve as a cell-free dressing, has been described (WO 01/41820).

 Chitosan is a biodegradable natural fiber, obtained after deacetylation of chitin. Chitin is a biopolymer of high molecular weight, non-toxic and biodegradable, and it is, after cellulose, the most widespread polysaccharide in nature. Chitin consists of a linear chain, the structure of which contains linked P-1,4 monomers of glucosamine (GIcN) and N-acetyl-glucosamine (GIcNAc) type. The extraction of chitin from the shell of crustaceans caught in the open Arabian Sea (a fleet of 3000 fishing boats collect 10 to 30 tonnes of shell each day) is done by chemical means, then the transformation by deacetylation of the chitin in

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 soda makes it possible to obtain chitosan. Chitosan is therefore obtained by removing enough acetyl group (CH3CO-) to allow the macromolecule to be soluble in most dilute acids. This operation, called deacetylation, releases the amino groups and gives the chitosan a cationic nature at pH # 7.

 Due to its intrinsic chemical structure, a chitosan-based biomaterial is perfectly compatible with tissues, and its high biological tolerance has been known for a long time (it has no inflammatory or allergic reaction after transplantation, nor any antigenic character). Chitosan is completely biodegradable, enzymes such as lysozyme present in wounds split this polymer into oligomers which are then eliminated by natural metabolism.

 Due to their biocompatibility with the tissues of the human body and their healing property, chitin and its deacetylated derivatives have demonstrated their effectiveness for all forms of dressings: articular skin, corneal dressing, sutures in surgery which are absorbed naturally after healing, but also in bone repair or in dental surgery in implants or in the healing of gums. Mention may also be made, among the embodiments, of well-tolerated contact lenses. Since 1987, a partially deacetylated chitin-based artificial skin, Beschitine, has been manufactured in Japan and is sold today in many European countries, in pharmacies. This skin is in the form of a film to be spread, only once, on the wounds, this dressing does not need to be renewed. Beschitine is gradually biodegraded by enzymes until the formation of a new epidermis. For insignificant first or slight second degree burns, this skin has a certain effectiveness, but not for severe deep second and third degree burns.

 The present invention aims to provide skin substitutes which are both bioactive and easy to use. It aims to

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 overcome certain difficulties and disadvantages of the dressings used to date and mentioned above. The inventors have in fact demonstrated that it is possible to cultivate skin cells on a bioresorbable matrix based on chitosan poured into the bottom of a culture vessel. The sheets of cells adhering to such a matrix have, compared to the cell sheets of the prior art, a certain number of advantages. Indeed, it is possible to detach these sheets from the culture container without using an enzymatic treatment, by a simple mechanical detachment of the matrix based on chitosan (FIG. 3). This avoids damage to the cells, which is inevitable during enzymatic treatment. In addition, the cell sheet thus obtained is much more resistant and does not need to be transferred to a gauze and to be stapled there. Significant advantages of the skin substitutes of the invention are linked to the properties of chitosan. Indeed, chitosan has an excellent oxygen permeability which is necessary to allow good viability of the cells in contact with the support, once the substitute is placed on the wound. It has a healing power and therefore promotes cell regeneration (biostimulating activity on the reconstitution of tissues). In addition, chitosan is an antiseptic and can therefore protect tissues from microbial (bacteriostatic) infections. Chitosan is also a particularly effective moisturizing agent, which has a double advantage: providing water and avoiding dehydration. This hydrating power also has the advantage of persisting over time.

 In addition, depending on the chitosan selected and the post-treatment carried out, it is possible to obtain a matrix whose surface is able to promote the adhesion and proliferation of human keratinocytes, or even to increase their clonogenic power and their mitotic index. .

 Compared to acellular dressings made of a chitosan film, such as Beschitine presented above, the skin substitutes of the invention have advantages linked to the presence of the cells cultured on their surface or within the matrix, in the case it is

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 porous. The skin substitutes of the invention, unlike the acellular dressings of the prior art, are therefore bioactive. In fact, the cells included in these substitutes, whether they are fibroblasts or keratinocytes, and whether they are allogenic or autologous, synthesize growth factors which will make it possible to improve the efficiency of healing, in particular by terms of speed and esthetics of closed wounds. In addition, the association between a matrix - consisting mainly of a chitosan-type polysaccharide - and differentiated keratinocytes and / or fibroblasts, presents a unique synergy in favor of re-epithelization and optimal cell regeneration, since the matrix alone already has a healing power.

 The invention therefore relates firstly, to a skin substitute consisting of a cell sheet of keratinocytes and / or fibroblasts, adhering to a bioresorbable matrix based on chitosan.

 By matrix based on chitosan is meant here a polymeric or copolymeric matrix comprising between 60 and 100% of chitosan, the other optional constituents being chosen in particular to optimize the mechanical properties (elasticity, flexibility, porosity, ...) of the matrix .

In the rest of the text, the matrix based on chitosan will also be designated by the terms matrix of chitosan, film (based) of chitosan, membrane (based) of chitosan, or foam (based) of chitosan. The terms film or membrane rather designate a crosslinked chitosan gel obtained by drying an aqueous solution comprising chitosan, according to a process described below, while the foam is obtained by lyophylization. By definition, a film or a membrane is therefore denser than a foam. The term matrix covers both the dense form and the foam.

 In a skin substitute of the invention, the cell sheet preferably comprises several layers of cells. These skin substitutes constitute an equivalent epidermis when the cultured cells are keratinocytes obtained after dermo-epidermal dissociation and

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 grown on a feeder undercoat, or an equivalent dermis when the cultured cells are differentiated fibroblasts. It is also possible to produce the skin substitutes of the invention, by inoculating dissociated keratinocytes on the equivalent dermis described above.

 In the skin substitutes of the invention, the cell sheet of the invention can also contain stem cells. It may indeed be advantageous to cultivate stem cells in vitro, and to direct their differentiation towards keratinocytes.

 This can be done either by using embryonic stem cells, in particular by the techniques developed by Bagutti and aL on mouse embryonic stem cells (Bagutti, Wobus et al.

1996) or on murine adult stem cells of the type used by the team of Yann Barrandon, using stem cells contained in the hair follicles (Oshima, Rochat et al. 2001).

 As the differentiation of stem cells into keratinocytes is not simultaneous for all cells, a mixed culture of stem cells and keratinocytes is thus obtained, which constitutes an equivalent epidermis having a good mitotic index. This technique makes it possible to envisage constituting equivalent epidermis with a view to allogeneic grafts, in particular for making allogeneic culture epidermis of burn victims awaiting autologous grafts after culture of their own cells. This kind of equivalent epidermis is certainly preferable to the skin of corpses used to temporarily cover burn victims, in particular because the cultured cells certainly release more growth factors than the skins of corpses.

 The use of stem cells as a source of keratinocytes also makes it possible to envisage the unlimited obtaining of grafted reconstituted epidermis without risk of rejection. Indeed, these cells have two exceptional characteristics: (i) they are immortal without being immortalized. This approach therefore makes it possible to obtain keratinocytes,

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 unlimited, from undifferentiated ES cells; (ii) these cells can be easily manipulated by transfection or homologous recombination.

In order for these cells to persist in a recipient patient, modifications of certain loci, such as that of the genes of the major histocompatibility complex (MHC), which play a major role in the recognition of foreign cells by the immune system, would make it possible to create immunotolerant universal pluripotent lines (Milner and Campbell 1992). Other gene modifications could be envisaged on these cells, such as, for example, the inactivation of viral receptor genes for transplants in patients suffering from chronic viral infection or the expression of repair genes. The problems inherent in primary cultures of keratinocytes (low quantity of cells, ineffective transfection, allograft problems) could therefore be avoided by this technique. Thus, treatment centers for burn victims could routinely have efficient and easy-to-use skin substitutes for temporary or permanent allogenic transplants.

 These allogeneic culture epidermis could also be used for the treatment of chronic wounds resulting from venous pathologies or pathologies linked to scarring defects, which require bioactive dressings gradually and durably releasing all the necessary growth factors, at physiological concentrations.

 The matrix chitosan in the skin substitutes of the invention preferably has a molecular mass of between 100,000 and 500,000 daltons, and an average deacetylation degree of between 50 and 100%, preferably between 70 and 90% (in reference to chitin whose deacetylation degree is 0%). The degree of deacetylation will in particular be chosen by a person skilled in the art according to the behavior and the cellular nature which he wishes to cause to grow on the surface or within the matrix of the skin substitute (Chatelet, Damour et al. 2001). Degree

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 deacetylation may also be chosen by a person skilled in the art according to the duration of resorption that he wishes for the matrix of the skin substitute, which may depend on the applications. In fact, the resorption will be faster the lower the degree of deacetylation. Thus, chitin and chitosan with a deacetylation degree of less than 66% are very quickly degraded (in less than three weeks) (Tomihata and Ikada 1997).

 In the skin substitutes of the invention, the chitosan matrix is preferably in the form of a three-dimensional hydrated membrane in the form of a crosslinked gel. This three-dimensionality allows in particular a good swelling power of the membrane, due to the easy incorporation of water molecules within the network. This water permeability, which results from the three-dimensional cross-linked structure of the membrane, also makes it possible to maintain a humidity level comprised in an optimal therapeutic window, after implantation of the skin substitute on the patient, and therefore has the advantage avoid dehydration. The matrix can also comprise polyvinyl alcohol or polyvinyl alcohol (PVA), in a proportion preferably between 10 and 40%, and / or glycerol.

 According to the invention, the membrane can be presented either in the form of a dense hydrated film, or as a hydrated porous foam, which can be colonized by cells. A preferred dermo-epidermal substitute of the invention therefore consists of a foam based on chitosan, colonized by fibroblasts, and carrying on its surface a sheet of keratinocytes. In this case, the porosity also allows better elimination of the liquids exuded by the wounds, after implantation of the skin substitute on the patients.

 According to a particular embodiment of the skin substitutes of the invention, the chitosan matrix has a thickness of between 0.1 and 2 mm, preferably between 0.3 and 1 mm.

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 According to a preferred embodiment of the invention, the chitosan matrix of the invention is imbibed by one or more growth factor (s) intended to promote healing, and / or one or more antibiotics. This property derives intrinsically from the process for obtaining the skin substitutes of the invention, described below, since these growth factors and antibiotic (s) are present in the cell culture medium, which differs depending on the inoculated cells (keratinocytes , fibroblasts, or stem cells), and in which the dermal substrate is balanced. By way of nonlimiting examples of growth factors that can be used, mention may be made of human EGF (Epidermal Growth Factor) of recombinant origin, triiodotyronine and cholera toxin (Vibriocholerae) of bacterial origin. We can also use the KGF (Keratinocyte Growth Factor) and FGF (Fibroblast Growth Factor) humans. The skin substitutes of the invention can also be imbibed with hormones and corticosteroids, such as insulin and hydrocortisone. Mention may be made, as non-limiting antibiotics, of gentamicin (Geomicine), amphetericin B (Fungysone), penicillin and streptomycin.

 The skin substitutes of the invention can be conditioned or packaged to ensure their preservation in good conditions, in particular to ensure their transport. Packaging methods are described in particular in patent application EP 1 085 081. According to a preferred packaging method, the skin substitutes of the invention are packaged in an individual sealed tray comprising gelled nutritive medium and a sterile mixture of approximately 95% air and 5% C02.

 The bioresorbable matrix based on chitosan which is included in the skin substitutes of the invention is preferably entirely biodegradable. Its absorption in vivo takes place by enzymatic degradation, and not by chemical hydrolysis.

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 The present invention also relates to a cell culture support for the production of skin substitutes; these supports comprise at least one matrix based on chitosan as described above, the surface of which is greater than or equal to 15 cm 2.

 Preferably, the chitosan of the matrix of culture supports according to the invention has a degree of deacetylation of between 50 and 100%, more preferably between 70 and 90%. Its molecular weight is preferably between 100,000 and 500,000 daltons.

 According to a preferred embodiment of the cell culture supports of the invention, the three-dimensional membrane of chitosan is stabilized in an aqueous medium. More preferably, this matrix (dense film) has good swelling power in aqueous media. This has the significant advantage of allowing the maintenance of the optimal humidity level at the level of the wounds. In a particular embodiment, the chitosan matrix is in the form of a porous foam, which can be obtained by the technique of freeze drying, or freeze-drying, described below.

As indicated above, such a foam has the advantage of being able to be colonized by cells, allowing the production of equivalent dermal models or dermo-epidermal models. This also has the advantage of allowing the evacuation of liquids exuded by the wounds.

 According to a particular embodiment of the cell culture supports of the invention, the chitosan matrix also comprises polyvinyl alcohol (PVA). The matrix of such supports preferably comprises between 60 and 90% of chitosan and between 10 and 40% of polyvinyl alcohol. The polyvinyl alcohol included in the matrix of cell culture supports of the invention is preferably cold-soluble and has a degree of hydrolysis which is preferably between 85 and 90%. An important parameter of the polyvinyl alcohol used in the cell culture supports of the invention is its viscosity, which is preferably between 20 and 30 centipoises at 20 ° C. for a solution at 4% by weight.

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 In the cell culture supports of the invention, the chitosan-based matrix preferably has a thickness of between 0.1 and 2 mm, more preferably between 0.3 and 1 mm. According to a preferred embodiment of these supports, the non-porous chitosan matrix is transparent, which makes it possible to have a transparent culture support allowing precise monitoring of the progress of cell culture.

 In the cell culture supports described above, the chitosan film is preferably adherent to the bottom of a solid support, from which it is detachable by mechanical action. The solid support in question preferably has the property of being able to be opened from above; nonlimiting examples of supports which can be used are peelable bottles such as those sold for example by the firm TPP (Techno Plastic Producs AG, Switzerland) under the references 90551 and 90651, or Petri dishes of any size greater than 15 cm 2 and of any geometry. In the cell culture supports of the invention, the chitosan-based matrix is preferably bioresorbable. As mentioned above, the degradation of chitosan in vivo takes place by enzymatic degradation and not by chemical hydrolysis.

 The inventors have identified a chitosan which is particularly suitable for constituting cell culture supports according to the invention: it is chitosan extracted from squid feathers, marketed for example by the company France Chitine (Marseille, France). Other chitosans which can be used in the context of the present invention are, for example, the chitosan of fungal origin sold by Kitozyme (Belgium), and the chitosan of animal origin and of GMP quality sold by Pronova (Canada). Of course, these sources of chitosan are given by way of illustration, and other chitosans can be used to carry out the invention, such as, for example, synthetic chitosans.

 Another aspect of the invention is a culture of adherent cells, chosen from a group comprising keratinocytes,

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 fibroblasts and stem cells, on a cell culture support as described above. Such a cell culture, when the chitosan matrix has been poured into the bottom of a culture flask, can be transported easily, for example by filling said flask with culture medium, optionally supplemented with hepatis.

 The invention also relates to a method for producing a cell culture support such as those described above, comprising the following steps: (a) dissolving chitosan in an aqueous solution of dilute acid, (b) pouring this solution homogenized in a culture vessel with a surface area greater than or equal to 15 cm2, (c) dry until a solid matrix is obtained, or lyophilized until a porous foam is obtained, and (d) post-treat said matrix or said foam with a basic solution, then removing the excess unreacted base; the salts possibly formed and trapped in the matrix (ammonium maleate for example), are also eliminated, at least partially, at this stage; (e) as necessary, equilibrating said matrix by rinsing with the culture medium which can be used for the cells which it is desired to cultivate.

 In a preferred implementation of this process, the solution prepared in step (a) comprises between 1 and 5% of chitosan, preferably between 1.5 and 3%. The dilute acid solution may for example be a solution of maleic acid, or of another acid, preferably with a carbon chain greater than C2 or C3, for example acetic, succinic or glutamic acid. It is also possible to mix these different acids, or to use other acids. The total concentration of acid in this solution is preferably between 0.09 and 0.27 mol / L. The pH of the

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 solution prepared in step (a) is for example between 1 and 5, preferably between 2 and 4.

 The solution prepared in step (a) may also contain polyvinyl alcohol (PVA), preferably in an amount such that the matrix within the support finally obtained contains between 10 and 40% of polyvinyl alcohol, preferably approximately 20%. weight relative to the weight of chitosan. For this, use between 0.2 and 1 g of PVA in 100 ml of film-forming solution to be poured (ie 0.2 to 1% by weight). This solution can also contain a third element, which will be at least partially eliminated during the post-treatment stage (d) and subsequent rinsing stages (e). In a preferred implementation of the above methods, the solution prepared in step (a) is a three-phase solution which contains between 0.4 and 1.2% glycerol in 100 ml of film-forming solution to be poured, chitosan and PVA, preferably in the proportions indicated above.

 In an implementation of the methods of the invention, the surface of the culture container is between 100 and 200 cm 2, preferably between 115 and 150 cm 2. Containers which can be used in these methods are, for example, vials of 150 cm 2, or 115 cm 2 flasks, the latter making it possible to obtain a culture support of rectangular geometry. In a preferred implementation of the methods of the invention, the container is a peelable bottle or the lower part of such a bottle before welding of the peelable film. In fact, it may be advantageous, in a production line for cell culture supports as described above, to pour the solution prepared in step (a) of the above process into the bottom of a bottle, the part of which is still open, which makes it easier to obtain a homogeneous surface and faster and more efficient drying. In such a production line, the container lid, which in the case of a peelable bottle, consists of a film of flexible plastic film glued to the edges of the vertical walls, is added after the steps of pouring and drying or

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 lyophilization (b) and (c) above and even optionally after step (d; of post-processing of the matrix.

 In the processes for producing supports as mentioned above, the drying step (c) can be carried out by evaporation under air flow, at a temperature between 15 and 50 ° C., preferably between 20 and 40 ° C. This step can also be carried out by lyophylization at low temperature between -40 ° C. and 0 ° C., preferably between -30 ° C. and -20 ° C., according to the freeze drying technique described by the team of J. Chen (Ma, Wang and al. 2001), in order to obtain a porous foam which can be colonized by cells.

 The chitosan solution is prepared as in step (a), with or without the addition of PVA and / or glycerol, and in the presence of a pore-forming agent dissolved in the solution, such as sodium chloride, glucosE or sucrose. The mixture is then poured into the appropriate mold for the invention, immediately frozen at -28 ° C., and finally lyophilized in a freeze-dryer (FTSTM Systems Inc, USA) to give a porous sponge. The thickness of the dry chitosan sponge can be controlled by changing the amount of chitosan solution used.

 Stage (d) of post-treatment of the matrix in the processes: of production of the support of the invention aims in particular to make this matrix stable in aqueous solution. This step can be carried out by immersing the matrix in a basic solution, for example in an ammonium hydroxide or sodium hydroxide solution, the concentration of which is preferably between 0.2 and 1, (mol / L , or by simple contact of the matrix with ammonia vapors. This basic post-treatment step of the matrix has several advantages: - it neutralizes the acid residues contained in the matrix at the end of the step (c); - it allows stabilization of the matrix by ionic cross-linking of the chitosan chains; and

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 - It generates free amine functions which improve the adhesion of cells in culture and in particular of keratinocytes.

 After this treatment in basic medium, the matrix is stable and cannot dissolve in aqueous medium. It can therefore withstand prolonged storage in its hydrated form, under culture conditions, while preserving its integrity, in particular its mechanical characteristics.

 Before cell culture, the supports of the invention are subjected to an additional step (e), which consists in rinsing the matrix in an aqueous medium (if necessary, after a first rinsing in a 70% isopropanol solution), for example in PBS and / or culture medium, which allows: - to rebalance the pH of the matrix; - to release the basic residues; and, optionally, - partially salting out the water-soluble glycerol from step (a), the presence of which is not desired at a high concentration in the final product.

 If necessary, it is also possible to perfect the supports of the invention by adding a step (f) of treatment of the surface of the matrix to allow selective adhesion of certain cell types, by grafting to the surface of the chitosan film. , by chemical coupling, microbial adhesins, enzymes or active ingredients (such as heparin for example via its terminal aldehyde group), or functionalized polymers (amine function at the surface).

 An important aspect of the methods for producing supports according to the invention is that the supports obtained must be sterile. This can be achieved in different ways. In a preferred implementation of the methods of the invention, all of the compounds used to manufacture the matrix are sterilized and each of the steps (a) to (e) or, where appropriate, (a) to (f) mentioned above. above, is carried out under sterile conditions. Three main modes of sterilization can be used:

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 membrane filtration with a porosity of 0.22, sterilization by radiation, for example by y-radiation, or autoclaving. The autoclaving of aqueous solutions of polysaccharides causing oxidation of the products, it is necessary to deoxygenate a solution comprising chitosan before its passage in the autoclave. This is generally carried out by bubbling with a neutral gas, for example nitrogen. The different compounds of the solution of step (a) can be sterilized by one of these methods. For example, in the case of a matrix consisting of chitosan and PVA in the presence of glycerol, the chitosan and the PVA are y-sterilized and the glycerol in solution is filtered through 0.22 before introducing it into the aqueous solution dilute (sterile) acid from step (a) above. It is also possible to sterilize the chitosan solution produced in step (a), before pouring, by autoclaving it after having completely deoxygenated it by bubbling with nitrogen.

 In a complementary or alternative manner, the methods of production of supports mentioned above comprise a final sterilization step, which is preferably carried out by irradiation under p or y radiation, or by chemical decontamination in an alcoholic solution, for example in l isopropanol at 70%. In the case where this step is carried out by irradiation, the amount of radiation must however be limited, since it can lead to degradation of the components of the matrix. The doses used in the food industry, conventionally of the order of 25 Kilograys, cannot be used in the context of the present invention, unless starting from a chitosan having a molecular mass much higher than that indicated above, to hold account for degradation subsequent to irradiation. This stage preferably takes place after stage (d), or, alternatively, after stage (e) of rinsing or after stage (f) of modification by grafting of the support obtained in (d) or (e) .

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 Finally, it can be envisaged that sterilization is carried out at any intermediate stage of the production process, the subsequent stages then being carried out under sterile conditions.

Another type of cell culture support for the production of skin substitutes according to the invention comprises fibroblasts trapped in a crosslinked gel based on chitosan. The invention also relates to a process for obtaining such a support, as described in Example 3, and comprising the following steps: (a) dissolving chitosan in an aqueous solution of dilute acid, (b) homogenize the solution and adjust the pH between 6.5 and
7.5, (c) add individual fibroblasts to the solution, (d) pour this solution containing the fibroblasts into a culture vessel with a surface area greater than or equal to
15 cm2, (e) let the solution stand until cross-linking of the chitosan gel.

 The invention also relates to a method for obtaining a skin substitute, by in vitro culture of cells on a support such as those described above, or on a support capable of being obtained by one of the methods described above. , further comprising a mechanical step of detaching said skin substitutes from the culture flask. The cells used in this process could in particular be chosen from keratinocytes, fibroblasts and embryonic or adult stem cells. This process for obtaining a skin substitute can also include a step of conditioning the skin substitutes in individual sealed trays, on a bed of gelled nutritive medium, under a sterile atmosphere of a mixture of air and CO2.

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 Another aspect of the present invention is the use of a skin substitute such as those described above or such as those capable of being obtained by the methods seen above, for carrying out in vitro tests of cosmetic compositions and / or pharmaceuticals.

 A preferred use of the skin substitutes of the invention aims at the manufacture of grafts for the repair of skin lesions. These skin lesions can be either acute wounds (injuries and / or burns) or chronic wounds (especially leg ulcers and pressure sores). The skin substitutes according to the invention can also be used for the manufacture of grafts for the treatment of congenital nevi. Indeed, the operation of congenital naevi in young children most often leaves unsightly scars which can be more or less annoying depending on where they are. The use of skin substitutes according to the invention can make it possible to manufacture autologous grafts which may give a much better result during the operation of these nevi.

 The present invention also relates to methods of treating acute skin lesions (burns and / or injuries) or chronic (ulcers, bedsores, etc.). As mentioned above, skin substitutes of the invention, in particular epidermal substitutes obtained from stem cells alone or in coculture with keratinocytes and / or fibroblasts, can advantageously be used in the case of severely burned and d venous ulcers, especially leg ulcers, to cover their wounds while waiting for an autologous transplant.

These skin substitutes could advantageously replace the skin of corpses currently used.

 Another aspect of the present invention is a method of treating congenital naevi comprising steps of taking a biopsy from the skin of the patient to be treated, a step of culturing these cells on a chitosan matrix in a cell culture support.

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 according to the invention, then the grafting of the autologous cutaneous substitute thus obtained, after excision of the skin constituting the nevus.

 The examples and figures below illustrate certain aspects of the implementation and of the advantage of the present invention, without however limiting its scope.

Caption of Figures Figure 1: of a chitosan membrane in a commercial peelable culture flask. Diagram of the process for preparing a culture vessel containing a chitosan membrane as obtained in Example 1. The criteria appearing in boxes correspond to the controls to be carried out before proceeding to the next step.

 Figure 2: the in vitro culture process leading to the skin substitute as obtained in Example 3. The criteria appearing in boxes correspond to the controls to be performed before proceeding to the next step.

Figure 3: illustrating one of the advantages of the process: easy mechanical detachment from the culture container of the skin substitute using sterile forceps (without enzyme and transfer) Figure 4: illustrating the adhesion and proliferation of adult keratinocytes (3 separate donors) on the surface of a film based on a crosslinked chitosan gel in a peelable (or peel-off) bottle of 150 cm2 (inoculation density: 10,000 cells / cm2) in comparison with a control surface (bottom of the peelable bottle):
A. after 2 days of seeding the keratinocytes on the feeder layer;
B. after 3 days of culture (for the 3 donors);
C. after 5 days of culture (for the 3 donors);

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D. between 7 and 14 days of culture for the same adult donor (KDo 62).

Example 1: culture supports in which the matrix based on a crosslinked chitosan sky is in the form of a dense film
The culture container used here is a peel-off culture bottle of the peel off type, sold by the company Techno Plastic Products AG (in Switzerland). This container can have a rectangular surface of 115 cm2 (reference 90551), or a polygonal surface of 150 cm2 (reference 90651).

 Initially, the inventors sought to optimize and define the specifications of the raw materials constituting the solution to be poured into the culture vessel which serves as a mold in the process.

The solution in question, called the film-forming solution, must allow the final film to have a certain number of characteristics, some of which are detailed below.

 The film-forming solution is prepared by dissolving, in 100 ml of aqueous solution of maleic acid (approximately 1.4% by weight; 0.12 mol / L), at pH around 3, the following raw materials: - 2 g of purified chitosan having a deacetylation degree between 80% and 90% and a molecular weight Mv between 150,000 and 300,000 daltons. It is possible to use chitosan of higher viscosity (Mv between 300,000 and 500,000 daltons), but it is then necessary to dissolve quantities twice as small for the same volume; - 0.5 g of synthetic polyvinylalcohol of USP quality (American pharmacopoeia), having a degree of hydrolysis of about 88% and a viscosity (at 20 C) between 20 and 30 CentiPoises; - 0.6 g of Ph Eur grade glycerol (European pharmacopoeia).

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 This proportion of each of the constituents in the complete solution to be poured has been optimized so as to obtain, after drying, a solid film of chitosan which in particular meets the following requirements: - have sufficient adhesiveness to the bottom of the mold (culture vessel), at the end of all the stages of implementation and throughout the duration of the subsequent in vitro culture process (epidermal cells); - have the ability to detach mechanically (using fine, sterile forceps) out of the peelable culture container, once the film has been combined with the cell sheet (at the end of the in vitro culture process) in one piece without tearing; - have good adhesiveness to the surface of the skin previously moistened when the graft is applied in vivo while preserving an optimal level of humidity in the wound.

 These requirements involved optimizing the chemical composition of the solution initially poured into the mold and, consequently, the final chemical composition of the film ready to receive the cells, so that this semi-finished product has flexibility and mechanical resistance (properties physico-mechanical) as well as an adequate and reproducible water content (swelling rate).

 After adding the 3 constituents of the acidulated solution, it was stirred for a few hours at room temperature before being autoclaved and then filtered through a 100 m filter. A volume (V = 50 ml) of sterile solution was then poured into a sterile peelable bottle (S = 115 cm2). This solution was then dried at room temperature under a continuous flow of air (in a sterile hood) until a dense dense film was obtained.

The thickness of this film, which influences the elastic and mechanical strength properties of the film, can be controlled by the amount of film-forming solution used per unit area. The drying time of the film is also dependent on this amount of solution poured per unit area.

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 The dense solid film obtained was then subjected to a post-treatment which consists in immersing it, for 1 hour at room temperature, in a solution (V = 60 ml) of ammonium hydroxide in an alcohol-water mixture (concentration of NH40H of about 1% by weight or 0.2 mol / L). The amount of base used and the contact time of this basic solution with the matrix have been optimized in order to effectively neutralize the acid functions contained within the film and to generate free amine functions in the polysaccharide chains, allowing ionic crosslinking of the gel. of chitosan and polyvinyl alcohol (PVA) constituting the matrix. After this treatment, the matrix is stable and can no longer dissolve in an aqueous medium: it can therefore be stored for an extended time, in its hydrated form, under the culture conditions (pH around 7) while preserving its physical properties. -mécaniques.

 Before use, the matrix must be rinsed in 70% isopropanol to neutralize this time the excess base used in the previous crosslinking step, then in phosphate buffer (pH = 7) to rebalance the pH within of the matrix (around 7).

 The dense film based on a crosslinked chitosan gel is finally balanced in culture medium, the nature and composition of which depend on the cellular nature (keratinocytes, fibroblasts or even stem cells) used in the subsequent in vitro culture process in to prepare an equivalent epidermis, an equivalent dermis or reconstructed skin. This equilibration step makes it possible to fix various active principles present in the culture medium such as antibiotics, growth factors, hormones or even corticoids. The matrix of chitosan soaked in medium promotes their effects.

 The preconditioning of this chitosan matrix in culture medium thus leads to the culture support ready to be brought into contact with the epidermal cells (see examples 4 or 7).

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 The different stages of the process described in this example are shown diagrammatically in FIG. 1, which also shows the checks carried out at the end of each stage.

 An example of cell proliferation is illustrated in Figure 4.

Example 2: Culture supports in which the chitosan matrix is in the form of a porous foam
In this example, the chitosan matrix is in the form of a porous foam. The culture vessel intended to serve as a support for this foam is identical to the support used in Example 1.

 Initially, the inventors sought to obtain a chitosan foam having the required specifications and characterized by a porosity allowing colonization of the cells (fibroblast type) within this three-dimensional porous matrix (and no longer cell growth exclusively at the surface of the dense chitosan film, which is the case in Example 1). This porous matrix filled with human fibroblasts constitutes an equivalent of dermis (example 5), used as a temporary dressing on wounds, or even an ideal support for reconstructing an epidermis (reconstructed skin: example 7).

 In this example, the film-forming solution to be poured into the culture vessel is prepared by dissolving chitosan (2 g) in 100 ml of aqueous solution of maleic acid (0.12 M) and optionally adding a proportion of PolyVinylAlcohol suitable for it the culture of fibroblasts (dermal cells). Instead of dissolving in the solution to be poured as the third component of glycerol (as is the case in Example 1), a water-soluble blowing agent - such as sodium chloride, glucose or sucrose - is dissolved (at a rate of 1 to 6% by weight) in the chitosan and / or PVA solution. The amount and nature of the blowing agent used influences the morphology of the foam obtained, namely the proportion, size and configuration of the pores generated during lyophilization.

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 The proportion of each of the constituents in the complete solution to be poured has also been optimized so as to obtain, after lyophilization, a chitosan foam meeting the same requirements as those mentioned in Example 1 but, moreover, with specific porosity criteria ( type, pore size).

 The complete solution once well homogenized with stirring is filtered through 100 microns, then sterilized after degassing with nitrogen by autoclave. The sterile solution is poured into the mold suitable for the invention (sterile culture container, here a peelable bottle). It is then immediately frozen at low temperature (-28 C). Once well frozen, the complete solution is then lyophilized in a freeze dryer to give a porous sponge of chitosan.

 The thickness of the dry chitosan sponge can be controlled by also changing the amount of complete solution poured per unit area. In this preparation protocol, the dry chitosan foam can have a controlled thickness of the order of 50 to 250 microns. The thickness of a normal human dermis is of the order of 0.5 to 2 mm depending on the age, sex and location of the sample. The goal of an artificial dermal substitute is to build a network that is conducive to the attachment and proliferation of dermal fibroblasts, rather for the purpose of a temporary dressing than for a permanent implant. Thus, the thickness of the artificial dermal substitute is generally much less than that of the actual human dermis.

 The post-treatment of the solid foam obtained, as well as the washes and equilibration in the culture medium (complete DMEM or Green) specific to the dermal cells (human fibroblasts) are carried out sterile as described in Example 1, and lead with the culture support ready to be brought into contact with the skin cells (see example 5).

 It is also conceivable to pour, over the porous chitosan foam thus prepared, a complete film-forming solution as prepared in Example 1, to prepare a dense film of

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 chitosan conducive to the adhesion of epidermal cells (see procedure in Example 1). This procedure then leads to the construction of a three-dimensional structure of the "bilayer" type, on the surface of which the reconstruction of an epidermis in vitro can be done to lead to a total reconstructed skin (see example 7).

EXAMPLE 3 Culture Supports in Which the Matrix Consists of a Chitosan Gel with Which Live Human Fibroblasts Are Associated (Dense Film)
In this example, the culture vessel in which the chitosan gel associated with fibroblasts (# discontinued) is used is a commercial petri dish (of any size) or a peelable culture flask as mentioned above.

 The dermal cells used in this example are human fibroblasts, which constitute the only cell population of the dermis (100% of the cells). A suspension of individualized fibroblasts is isolated from a human skin biopsy (operational waste), by dermo-epidermal dissociation and conventional enzymatic treatment (with trypsin). These cells can be cultivated several cycles but only used in the interval up to 7 to 8 cycles of "subculture" after the primary culture.

 In this example, the film-forming solution to be poured into the culture vessel is prepared by dissolving chitosan (between 3 and 5% by weight) in slightly acidic Green medium (and adding or not adding a proportion of PVA adapted to fibroblasts) .

 It is important, in this case, to readjust the pH value of this viscous solution made up of chitosan gel towards neutrality (pH = 7) before associating the dermal cells with it. Instead of dissolving in the casting solution another component (as is the case in Examples 1 and 2), a suspension in DMEM medium complete with

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 individualized fibroblasts just harvested by treatment with trypsin, is mixed with chitosan gel.

 In this example, the crosslinking of the chitosan gel containing the fibroblasts takes place once the two elements (chitosan gel and fibroblasts) are brought into contact with one another (without requiring an evaporation step followed by the step neutralization in basic medium). The crosslinking of the chitosan gel and the organization of the fibroblasts within the crosslinked chitosan gel (3D network) can cause a contraction of the gel (reduction of the size of the support, of the membrane) which is stabilized after a few days of culture in immersion.

 In this example, the membrane made up of chitosan gel in association with human fibroblasts is from the start placed in immersion in the culture medium (from Green). Human fibroblasts once trapped within this network no longer have the ability to proliferate, but they are still alive and therefore still secrete growth factors of interest in Green's environment: they are said to be quiescent.

Thus, an equivalent dermis can be obtained which can be used either as a temporary dressing (allogenic dermal substitute) on wounds, or as an ideal support for reconstructing an epidermis (reconstructed skin: example 6).

 The thickness of the equivalent dermis can be controlled by also changing the amount of solution (chitosan + fibroblasts) poured per unit area. In this preparation procedure, the support can have a thickness of the order of 50 to 250 microns.

 The different steps of this protocol are summarized in Figure 2.

EXAMPLE 4 Skin Substitutes Consisting of Epidermal Cells Cultivated on a Dense Film of Chitosan
In this example, the skin substitutes consist of a chitosan film as prepared in Example 1, covered with a sheet

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 multi-layer epidermis. In this case, the chitosan matrix was packaged in Rheinwald and Green medium (containing EGF, cholera toxin, insulin, hydrocortisone, triiodothyronine as well as two antibiotics: Géomycine and fungizone).

 The epidermal cells used in this example are keratinocytes, which constitute the main epidermal cell population (95% of the cells). A suspension of epidermal cells is obtained from a biopsy of human skin (operational waste), by dermo-epidermal dissociation and enzymatic treatment. This suspension contains keratinocytes and melanocytes which can grow and therefore be amplified specifically (100% of cells of a single type), thanks to a selective medium.

 The day before the keratinocytes are sown, a nourishing underlay made up of 3T3 fibroblastic cells, either irradiated or mytomycinated and, consequently, in growth arrest, is inoculated on the chitosan matrix, balanced in Rheinwald and Green medium. The inoculation density of these 3T3 growth arresters is 20,000 cells / cm2 and the culture is carried out under an atmosphere humidified with 5% CO 2 at 37 C.

 In the 24 hours following the inoculation of 3T3, a suspension of dissociated keratinocytes is then inoculated on the feeder layer, at a rate of 4000 to 10,000 cells / cm2. Co-culture according to the Rheinwald and Green technique is continued until reaching confluence plus 3 days. During the last days of culture, a differentiation process is initiated, which results in the formation of a multilayer epithelial sheet (3 to 6 layers of keratinocytes), without however reaching the formation of the superficial corneal layer made up of dead anucleated cells, flattened, called corneocytes, which form an impermeable and protective barrier.

 After this in vitro culture, the chitosan matrix covered with the multilayer cell sheet is then mechanically detached using

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 fine sterile forceps. This recovery of the skin substitute from the culture vessel therefore does not require any enzymatic treatment (at the dispase) such as that practiced to produce the commercial autologous epidermal sheets. In addition, the resistance of the cell sheet anchored on the chitosan matrix after simple mechanical detachment is adequate. The preparation of the skin substitute of the invention therefore does not require a transfer of the sheet to a sterile gauze using expensive surgical staples as practiced to produce commercial autologous epidermal sheets.

 After recovering the sheet on film from the bottle, it is rinsed in MEM and then deposited on a bed of MEM culture medium gelled in a 2% Low Melting Point agarose solution, inside trays individualized. The skin substitutes of the invention are then placed under a sterile atmosphere consisting of an air / COz 95/5 mixture in these individual trays, before these are sterile and hermetically sealed.

 Once the skin substitutes are packaged, they are finally transported at low temperatures to the health center (place of transplantation) within 24 to 48 hours.

EXAMPLE 5 Skin Substitutes Consisting of Dermal Cells Cultivated Within the Porous Matrix Based on Chitosan
In this example, the skin substitutes consist of a tell chitosan foam that prepared in Example 2, colonized by living and differentiated human fibroblasts. In this example, the chitosan foam is conditioned either in culture medium suitable for human fibroblasts (DMEM containing 10% of serum such as FCS as well as antibiotics) if one wishes to produce an equivalent dermis for temporary use, or in Green's medium if the dermal substrate is later used as a support to reconstruct an epidermis.

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 The dermal cells used in this example are the fibroblasts which constitute the only cell population of the dermis (100% of the cells). A fibroblast suspension is isolated from a human skin biopsy (operational waste), by dermo-epidermal dissociation and conventional enzymatic treatment (with trypsin). These cells can be used up to 7 to 8 cycles of "subculture" after the primary culture.

 Once the foam is balanced in DMEM-10% FBS medium, the dissociated fibroblasts are inoculated therein, at an inoculation density around 20,000 cells / cm2. The culture is carried out under an atmosphere humidified with 5% of CO 2 at 37 C.

 The culture is continued until complete colonization of the pore volume constituting the chitosan foam. During the last days of culture, a process of differentiating the fibroblasts associated with the chitosan matrix is also initiated.

 After this in vitro culture, the chitosan foam colonized by differentiated fibroblasts is then mechanically detached using fine sterile forceps. The steps for recovering the skin substitute from the culture container, for conditioning and transporting this dermal substitute (or equivalent dermis) are completely similar to those described for the epidermal substitute (equivalent epidermis) in Example 4.

EXAMPLE 6 Skin Substitutes Consisting of Epidermal Cells Cultivated on an Equivalent Dermis Resulting from the Association of a Chitosan Gel and Human Fibroblasts in the Form of a Dense Film
In this example, the skin substitutes consist of a support consisting of a chitosan gel associated with fibroblasts as prepared in Example 3, covered with a reconstructed epidermis. In this case, the support, equivalent to dermis, is preconditioned in medium

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 Rheinwald and Green (containing EGF, cholera toxin, insulin, hydrocortisone, triiodothyronine as well as two antibiotics: Geomycin and fungizone).

 The epidermal cells used in this example are the keratinocytes, which constitute the main epidermal cell population (95% of the cells). A suspension of epidermal cells is obtained from a biopsy of human skin (operational waste), by dermo-epidermal dissociation and enzymatic treatment. This suspension contains keratinocytes and melanocytes which can grow and therefore be amplified specifically (100% of cells of a single type), thanks to a selective medium.

 As soon as the chitosan gel system associated with fibroblasts is stabilized after a few days in Green's medium, a suspension of dissociated keratinocytes (just harvested by treatment with trypsin) is then inoculated between 4000 and 10,000 cells / cm 2 at surface of the dermal substrate. The proliferation of epidermal cells occurs for 7 days in an immersion phase (Green medium).

 Then, for the next 7 days, the culture is placed in emersion, so as to induce the process of differentiation of the multilayer epithelial sheet, until the formation of the superficial stratum corneum consisting of the corneocytes (dead anucleated, flattened cells, which form an impermeable and protective barrier).

 After this in vitro culture, the reconstructed skin is then mechanically detached using fine sterile forceps.

 The steps for recovering the skin substitute from the culture container, for conditioning and transporting this dermal substitute (or equivalent dermis) are completely similar to those described for the epidermal substitute (equivalent epidermis) in Example 4.

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EXAMPLE 7 Skin Substitutes Consisting of Epidermal and Dermal Cells Cultured on a Porous Matrix Based on Chitosan
The skin substitutes here consist of a simple chitosan foam, or of a three-dimensional structure of the "bilayer" type, constituted below a chitosan foam and above a dense film of chitosan as it was described in Example 2. In this case, the skin substitutes as reconstructed skin come from the seeding of dissociated keratinocytes, on an equivalent dermis such as that obtained this time in Example 5.

 Once the foam is well colonized with human fibroblasts in Green's medium, a suspension of dissociated keratinocytes (just harvested by treatment with trypsin) is then inoculated between 4000 and 10,000 cells / cm2 on the surface of the dermal substrate. The proliferation of epidermal cells occurs for 7 days in an immersion phase (Green medium).

 Then, for the next 7 days, the culture is placed in emersion, so as to induce the process of differentiation of the multilayer epithelial sheet until the formation of the superficial stratum corneum consisting of corneocytes (anucleated, flattened dead cells, which form a waterproof and protective barrier).

 After this in vitro culture, the reconstructed skin is then mechanically detached using fine sterile forceps.

 The steps for recovering the skin substitute from the culture container, for conditioning and transporting this dermal substitute (or equivalent dermis) are completely similar to those described for the epidermal substitute (equivalent epidermis) in Example 4.

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 BIBLIOGRAPHY Bagutti, C., A. M. Wobus, et al. (1996). "Differentiation of embryonal stem cells into keratinocytes: of wild-type and beta 1 integrin-deficient cells." Dev Biol 179 (1): 184-96.

Chatelet, C., 0. Damour, et al. (2001). "Influence of the degree of acetylation on some biological properties of chitosan films."
Biomaterials 22 (3): 261-8.

Ma, J., H. Wang, et al. (2001). "A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a scaffold of human neofetal dermal fibroblasts."
Biomaterials 22 (4): 331-6.

Milner, CM and RD Campbell (1992). "Genes, genes and more genes in the human major histocompatibility complex." Bioessays 14 (8):
71.

Oshima, H., A. Rochat, et al. (2001). "Morphogenesis and renewal of hair follicles from adult multipotent stem cells." Cell 104 (2): 233-45.

Tomihata, K. and Y. Ikada (1997). "In vitro and in vivo degradation of films of chitin and its deacetylated derivatives." Biomaterials 18 (7): 567-75.

Claims (33)

 CLAIMS 1. Skin substitute consisting of a cell sheet of keratinocytes and / or fibroblasts, adhering to a three-dimensional bioresorbable matrix based on chitosan. 2. Skin substitute according to claim 1, in which the cell sheet comprises several layers of cells. 3. Skin substitute according to claim 1 or 2, wherein the cell sheet further comprises stem cells. 4. Skin substitute according to claim 3, in which the stem cells can be of embryonic or adult origin. 5. Skin substitute according to any one of claims 1 to 4, in which the chitosan-based matrix is in the form of a dense hydrated film or of a hydrated porous foam. 6. Skin substitute according to any one of claims 1 to 5, in which the chitosan-based matrix also comprises polyvinyl alcohol and / or glycerol. 7. Skin substitute according to any one of claims 1 to 6, in which the chitosan-based matrix is soaked with one or more growth factor (s) intended to promote healing and / or one or more antibiotics ( s). 8. Skin substitute according to claims 1 to 7, characterized in that it is packaged in a sealed tray containing gelled nutritive medium and a sterile mixture of approximately 95% air and 5% CO2. <Desc / Clms Page number 34> 9. Cell culture support for the production of skin substitutes according to any one of claims 1 to 8, in which the culture surface consists of a three-dimensional matrix based on chitosan, the surface of which is greater than or equal to 15 cm2. 10. Cell culture support according to claim 9, in which the chitosan of the matrix has a degree of deacetylation of between 50 and 100%, preferably between 70 and 90% and has a molecular mass of between 100,000 and 500,000 Daltons. . 11. Cell culture support according to any one of claims 9 to 10, in which the chitosan-based matrix is in the form of a porous foam. 12. Cell culture support according to any one of claims 9 to 11, in which the chitosan-based matrix also comprises polyvinyl alcohol and / or glycerol. 13. Cell culture support according to any one of claims 8 to 12, in which the chitosan-based matrix has a thickness of between 0.1 and 2 mm, preferably between 0.3 and 1 mm. 14. Cell culture support according to any one of claims 9 to 13, characterized in that the chitosan-based matrix adheres to the bottom of a solid support outside of which it is detachable by mechanical action. 15. Cell culture support according to claim 14, characterized in that the solid support is a peelable bottle or a Petri dish. 16. Culture of adherent cells chosen from a group comprising keratinocytes, fibroblasts and stem cells, on a <Desc / Clms Page number 35>  cell culture support according to any one of claims 9 to 15. 17. A method of producing a cell culture support according to any one of claims 9 to 15, comprising the following steps: (a) dissolving chitosan in an aqueous solution of dilute acid, (b) pouring this homogenized solution in a culture vessel with a surface area greater than or equal to 15 cm 2, (c) dry until a solid matrix is obtained or lyophilized until a porous foam is obtained, and (d) post-treat said matrix or said foam with a basic solution, then remove the excess base. 18. The method of claim 17, further comprising a step (e) of washing the matrix or the chitosan-based foam with a buffered solution and / or with culture medium. 19. The method of claim 17, comprising an additional step of treatment by grafting or coupling of active ingredients on the matrix obtained in (d) or (e) to allow selective adhesion of certain cell types. 20. Method according to any one of claims 17 to 19, in which the solution prepared in step (a) comprises between 1 and 5% of chitosan, preferably between 1.5 and 3% by weight and is at a pH between 1 and 5, preferably between 2 and 4. 21. Method according to any one of claims 17 to 20, in which the solution prepared in step (a) contains maleic acid and / or acetic acid, and / or succinic acid, and / or acid <Desc / Clms Page number 36>  glutamic, in a concentration such that the final acid concentration is between 0.09 and 0.27 mol / L. 22. Method according to any one of claims 17 to 21, in which the solution prepared in step (a) additionally contains 0.2 to 2% by weight of glycerol and / or 0.2 to 1% by weight polyvinyl alcohol. 23. The method of claim 17, wherein the surface of the container is between 100 cm2 and 200 cm2. 24. Method according to any one of claims 17 to 23, in which the container is a peelable bottle, or the lower part of such a bottle. 25. The method of claim 17, wherein step (c) is carried out by evaporation under air flow, at a temperature between 15 and 50 C, preferably between 20 and 40 C, or by lyophylization at low temperature between -40 C and 0 C, preferably between -30 C and -20 C. 26. The method of claim 17, wherein step (d) is carried out by immersing the matrix in a basic solution or by simple contact of the matrix with ammonia vapors. 27. The method of claim 26, wherein the basic solution is a solution of ammonium hydroxide or sodium hydroxide, the concentration of which is preferably between 0.2 and 1.0 mol / L. 28. The method of claim 17, further comprising a sterilization step, which is preferably carried out by irradiation under beta or gamma radiation, or by chemical decontamination in an alcoholic solution. <Desc / Clms Page number 37> 29. Process for obtaining a cell culture support for the production of skin substitutes, comprising the following steps: (a) dissolving chitosan in an aqueous solution of dilute acid, (b) homogenizing the solution and adjusting the pH between 6.5 and 7.5, (c) add individual fibroblasts to the solution, (d) pour this solution containing the fibroblasts into a culture vessel with a surface area greater than or equal to 15 cm 2, (e) leave the solution to stand until the matrix based on chitosan. 30. A process for obtaining a skin substitute, by culturing cells on a support according to any one of claims 9 to 15, or capable of being obtained by a process according to any one of claims 17 to 29, comprising a mechanical step of detaching the skin substitutes from the culture flask. 31. The method of claim 30, further comprising a step of packaging the skin substitutes in individual sealed trays, on a bed of gelled nutritive medium, under a sterile atmosphere of air / CO 2 mixture. 32. Use of a skin substitute according to any one of claims 1 to 8, or capable of being obtained by a process according to either of claims 30 and 31, for carrying out in vitro tests of cosmetic compositions and / or pharmaceuticals. 33. Use of a skin substitute according to any one of claims 1 to 8, or capable of being obtained by a process according to any one of claims 30 and 31, for the <Desc / Clms Page number 38>  manufacture of grafts for the repair of skin lesions, for the treatment of acute wounds, or for the treatment of chronic wounds, or for the treatment of congenital nevi.