Skin patch comprising a bioabsorbable skin substitute and relative production process

Abstract

The subject matter of the invention relates to a bioabsorbable skin substitute A) for ulcers and wounds, obtained by subjecting a composition A1) comprising, a cellular component a) in turn comprising fibroblasts, and a mixture b) comprising hyaluronic acid, fibrin and / or platelet-rich plasma gel (PRP), to a cryogenic procedure comprising the following steps: cooling at 4°C and maintaining the composition A1) at such a temperature for a time comprised between 1 and 2 hours; freezing at -20°C of the composition A1) and maintaining the same at this temperature for a time of at least 18 hours.

Description

[0001] TITLE: 'Skin patch comprising a bioabsorbable skin substitute and relative production process'

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The field of the present invention relates to skin substitutes. More specifically, the present invention relates to a skin patch comprising a bioabsorbable skin substitute and relative production process.

[0005] BACKGROUND ART

[0006] Some epithelial tissue lesions are referred to as 'difficult wounds,' which is a multiple concept intended to emphasise the characteristics of chronicity, lack of spontaneous closure and the presence of concomitant systemic biological factors which hinder normal tissue repair. A 'difficult wound' is defined as a lesion which, regardless of the underlying cause, is outside the normal processes and timeframe for resolution.

[0007] In the context of epithelial lesions, a chronic cutaneous ulcer (acronym CCU or CU for chronic ulcer) is defined as a lesion characterised by loss of substance, which persists in the absence of a tendency to spontaneous healing for more than 60 days (Westerhof ffl. Leg ulcers: diagnosis and treatment. 1993 Elsevier). The current prevalence of ulcers, including foot ulcers, can be estimated to be comprised between 0.18% and 0.32%, and between 0.06% and 1.0% for venous ulcers. Studies considering both open and closed ulcers estimate an overall prevalence in the order of 1-1.3 (The Alexander House Group -1992- Consensus Paper on Venous Leg Ulcer. Phlebology 7:48-58). The prevalence figures for vascular lesions are in the range of 1.8 to 3.05 per thousand, with increasing prevalence as age increases. In Western countries, it has been estimated that 10 per thousand of the adult population has been affected by a lower limb ulcer at least once in their lifetime (Margolis DJ, Bilker ffl, Santanna J, Baumgarten M. Venous leg ulcer: Mar;46(3):381-6.). The incidence is significantly higher in females, with a ratio of 3 to 1. Phlebostatic ulcers are among the most frequent, with a prevalence of 70-80%, followed by arterial ulcers (15-20%). An epidemiological survey conducted by Canonico et al. for the Osservatorio Geriatrico della Regione Campania Canonico et al. Prevalence of veins in an Italian elderly population (Angiology 1998; 49: 129 - 3 135), showed that 42.5% of males and 57.5% of females over 65 years of age are carriers of varicose disease of the lower limbs, and that 3% are carriers of skin ulcers. More recent analyses have also confirmed the epidemiological relevance of venous ulcers (Agale SV Chronic Leg Ulcers: Epidemiology, Aetiopathogenesis, and Management 2013 Ulcers 5 Volume 2013 Article ID 413604, http: Jdx.doi.org / lO.l 155 / 2013 / 413604).

[0008] If in hospitalised patients, pressure lesions develop with a prevalence ranging from 18% to 29% (A.I.S.Le.C. 2010), large, well-conducted studies are hard to find in the home care population. Forecasts by the World Health Organisation have estimated that the number of diabetics in 2025 will be more than 300 million with respect to the 120 million calculated in 1996. From this, it is easy to intuit the dimension assumed by this problem. In fact, it is estimated that about 15% of diabetics will experience a foot ulcer in their lifetime which will require medical attention. Although the diabetic population accounts for approximately 3% of the general population, more than 50% of all major amputations affect precisely diabetics.

[0009] The socio-economic impact of CUs is considerable. When assessing the costs of such a pathology, account was taken of expenses related to medication material, transport time and medical and nursing staff; other expenses incurred directly by the patients (private healthcare, adjuvant care, loss of working days, etc.) were not taken into account, as is the case for indirect expenses borne by the National Health Service (hospitalisations for surgery, infectious complications, haemorrhagic episodes, neoplastic degeneration, etc.) which would further increase the costs of this pathology (Rith-Najaran SJ et al. Identifying diabetic patients at high risk for lower extremity, amputation in a primary care setting. Diabetes Care 1992; 15:1386-9'). Anglo-Saxon studies on the cost of dressing material for the treatment of chronic ulcers indicate an expenditure of approximately £1,300 to £2,500 per patient in the UK, which corresponds to an annual cost of between £2 and £3 billion (Rippon etal The economic 30 impact of hard to heal leg ulcers Wounds 2007; Vol 3, N2; Posnett, J., Franks, P.J, (2008) The burden of chronic wounds in the UK. Nursing Times; 104: 3, 44 45). The impact of such a pathology on healthcare costs has long been known. For example, in Sweden (Baker, S., Stacy, M., Jopp-McKay, A., & Thompson, P. (1991). Epidemiology of chronic venous ulcer s.British Journal of Surgery, 78(7), 864-867) more than £120 million was spent annually, corresponding to about one percent of the national healthcare budget. In the United States, the authors Phillips et al. (Phillips T, 5 Stanton B, Provan A, Lew R. A study of the impact of leg ulcers on quality of life: financial, social and psychologic implications. J Am Acad Dermatol 1994, 31: 49-53) reported the loss of 2 million working days due to lower limb ulcers (about 2.5 million people would be affected). These considerable figures can be understood in light of the fact that 45% of skin ulcers result in immobility or a marked reduction in individual autonomy. All this without taking into account the deterioration of the quality of life. More recently, the estimated cost per venous ulcer patient in Germany was approximately € 9,500 (Purwis S. et al Cost-of Illness of chronic leg ulcers in Germany Int Wound 2010; 7: 97-102\ as well as extremely high costs for the healthcare budget continue to be reported (van Gent WB et al Management of venous ulcer disease; BMJ 2010; 341: c6045).

[0010] This picture is associated with the prospective involvement of an ever-increasing proportion of patients, both because of the increase in average life expectancy with the consequent increase in the most exposed age groups, and because of the increase in pathologies associated with ageing and / or peripheral vascular disease (e.g. diabetes), with a consequent ever-increasing proportion of lesions which will become chronic and / or refractory to the available therapies.

[0011] The consequence of all this is that chronic lower limb ulcers (CLLU acronym) constitute, and will continue to constitute in the coming years, one of the most difficult challenges of current medical research, both because of their significant epidemiological relevance, and because there is a large proportion of lesions which do not heal, or rather relapse, despite the patient being subjected to a correct therapeutic approach, with a consequent impact on treatment and management costs (hospitalisation, surgery, etc.) and social costs.

[0012] Traditional therapy for the treatment of ulcers involves, for example, compression bandaging, which only allows healing to be achieved in the long term; however, complete and faster healing is desirable in order to avoid complications, often capable of threatening the patient's life. Over the past 30 years, tissue engineering techniques have enabled the large-scale production of skin substitutes (acronym SS) (Varkey M at al J Funct Biomaterials 2015; 6: 547-563). Skin substitutes are classified on the basis of the Technology Assessment Program - Agency for Healthcare - Meriland USA - report Dec 12: into the following product types:

[0013] 1. products containing autologous or allogenic human cells;

[0014] 2. products made with human donor tissue;

[0015] 3. products derived from decellularized animal tissue;

[0016] 4. biosynthetic products.

[0017] Although these new therapeutic options constitute significant improvements over more traditional approaches, there is still a need to find a decisive cure for chronic ulcers.

[0018] For example, the product types 1-4 above have allowed an improvement in care capacity, without however satisfactorily solving the healthcare demand. It should be noted in this regard that CUs and consequently CLLUs fall into the category of pathologies which have 'unmet medical need': in this category, the European Medicines Agency (EMA) includes those pathologies whose current therapeutic approach is unable to ensure proper treatment for patients.

[0019] Due to the characteristics of the wound bed of chronic ulcers, the most suitable skin substitute for this type of lesion is product 1, containing autologous or allogenic human cells processed in GMP-authorised Cell Factories for the production of ATMPs (Advanced Therapies Medicinal Products), which fall under European Regulation 1394 / 2007.

[0020] This type of product also has a number of drawbacks. The causes which lead to a failure of healing CU and in particular CLLU are of various types such as: the size of the ulcers; the presence of bacterial species within the wound; the protracted nature of such ulcers over time (Slade B et al Phlebological rev 2015; 2:29-38).

[0021] In addition to this, the skin substitutes currently in use have a number of limitations in their use, most of which are due to the fact that they are not capable of ensuring a sufficient revascularisation and have a low adhesion to the wound bed, hampering skin tissue regeneration and thus wound healing. As far as chronic skin lesions are concerned, autologous skin substitutes, i.e., those made from skin samples of the patient to be treated, require relatively long production times and are therefore generally not available for the emergency treatment of lesions over a large body surface area (BSA). Furthermore, autologous skin substitutes are of little use in the treatment of patients suffering from dystrophic epidermolysis bullosa (DEB); moreover, the cells of patients with infectious diseases present serious difficulties for their processing in the pharmaceutical factories authorised to produce ATMPs, and so these patients are effectively precluded from access to this type of treatment (this occurs in over 70% of cases).

[0022] Allogenic skin substitutes, i.e., made from skin samples of different subjects (living or cadaver donors), processed in the Cell Factories, are instead very useful for the treatment of patients with extensive burns, with extensive skin loss, and can be used on infected wounds after thorough surgical cleaning of the wound bed.

[0023] Recently, several procedures have been proposed for making skin substitutes based on fibroblasts and keratinocytes as well as from other human cells (peripheral blood monocyte cells - PBMNC), for example those developed by:

[0024] 1. Organogenesis - Apligraft: is a produced formed from a skin transplant containing viable keratinocyte and fibroblast cells grafted onto a bovine collagen type 1 support.

[0025] 2. S&N - HP802-247: the product consists of expanded keratinocyte and fibroblast cells grafted onto a fibrin gel. The cells are irradiated and then subsequently frozen to stop the growth process thereof.

[0026] 3. CureXcell - Macrocure: is a product based on PBMNC cells concentrated by means of centrifugation and activated through a hypo-osmotic shock process (suspension of the cells in a solution with a low saline concentration). The product is injected directly at the site of the lesion: the proposed action mechanism is to stimulate tissue regeneration through the action of growth factors contained in the cells.

[0027] However, such procedures still have several disadvantages such as:

[0028] 1. Organogenesis - Apligraft. It is authorised on the US market as a medical device, but has not obtained approval on the European market due to the incorrect definition of the action mechanism of the cellular components. Moreover, the clinical evidence is judged by the US regulatory authority (FDA) to not meet treatment needs.

[0029] 2. S&N - HP802-247: after expansion, the cells undergo an irradiation process which destroys the alarmin proteins and growth factors within the fibroblasts. This results in a product which is safe, but without the regenerative capacity required for treating LCCs. During the execution of the phase 3 clinical trial in the US, it was found that the product did not meet the predetermined efficacy targets: the manufacturer's website published that the trial was suspended because the product did not show a significant clinical advantage with respect to the placebo treatment (httg: / 7www.smith- neptiew com / news-and-media / news / top-hne-resuits-oi-hp802-24 / -phase-3-siudv / ).

[0030] 3. CureXcell - Macrocure. The company carried out the phase 3 study for CureXcell for registration as an advanced therapy medicinal product (EMA study code - MC- 105) for the treatment of LCCs: the company admitted that the product did not meet the clinical efficacy endpoints and therefore the registration process is suspended.

[0031] The skin is the fundamental organ which separates the organism from the outside, and for this reason it possesses certain peculiarities at the molecular level which allow it to act as a physical barrier, to produce antimicrobial substances (at the level of the epidermis) and to carry out immunomodulatory functions (at the level of the dermis).

[0032] The repair processes in skin require the concurrence of several factors, among them hyaluronic acid, which is the most abundant molecule in both the dermis and the epidermis. The presence of hyaluronic acid is a necessary marker for the initial processes of healing and neoangiogenesis, as well as accompanying inflammatory phenomena necessary for reparative processes. The role of hyaluronic acid in the epidermis is critical, as it enables the differentiation of keratinocytes and is particularly abundant in the spinous and granular state of the epidermis. The absence of hyaluronic acid is not compatible with life, while its overproduction in the dermis is present in the shar-pei dog breed, while the longevity of the naked mole rat has recently been attributed to the amount of high-molecular-mass hyaluronic acid (Tian X, Azpurua J, Hine C, Vaidya A, 20 Myakishev-Rempel M, Ablaeva J, Mao Z, Nevo E, Gorbunova V, Seluanov A. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat. Nature. 2013 Jul 18;499(7458):346-9). From what has been reported, the topical use on the damaged epidermis of preparations containing both high molecular weight HA with moisturising and protective action appears to be scientifically tenable.

[0033] It has recently been observed that the biological action of HA fragments is strongly dependent on the size of the oligosaccharides. In fact, the functions of HA radically change depending on its size, sometimes producing opposite effects. Recently, it has been noted that the role of HA fragments is critical in reparative processes such as neoangiogenesis (Gao F, Yang CX, Mo ffl, Liu 10 YW, He YQ. Hyaluronan oligosaccharides are potential stimulators to angiogenesis via RHAMM mediated signal pathway in wound healing. Clin Invest Med. 2008;31(3): E106-16.) and epidermal repair (Gariboldi S, Palazzo M, Zanobbio L, Selleri S, Sommariva M, Sfondrini L, Cavicchini S, Balsari A, Rumio C. Low molecular weight hyaluronic acid increases the self-defense of skin 15 epithelium by induction of beta-defensin 2 via TLR2 and TLR4. J Immunol. 2008 Aug l;181(3):2103-10.\ as well as the presence of oligosaccharides in tissues during tissue repair processes is a natural phenomenon. HA oligosaccharides are produced by the tissue itself through mechanisms which are still poorly understood but involve both enzymatic (endogenous hyaluronidases) and physical (free radicals) digestion.

[0034] Despite this, dermal substitutes based on hyaluronic acid derivatives do not allow an appropriate treatment of LCC.

[0035] In addition, production processes of skin substitutes, which may also comprise fibroblast cells, are known to involve a cryopreservation phase. The latter, however, seems to be merely functional for the conservation of the skin substitute, as well as for the purpose of obtaining reserves to store. The temperature of the cryopreservation phase for the known processes is very low and lies in a range comprised between - 180°C and -80°C. These low temperatures require a higher degree of complexity in the production process, as well as higher operating costs. Often, in fact, the low temperatures are obtained by using liquid nitrogen.

[0036] Therefore, there is a need for epithelial tissue substitutes, in particular allogenic skin substitutes, which overcome the drawbacks of those known from the background art and / or have better clinical efficacy. In addition to this, there is the need to employ leaner or less complex production processes, and to favour an increase in the production of molecules capable of promoting the partial or total regeneration of the injured / wounded or ulcerated epithelium.

[0037] SUMMARY OF THE INVENTION

[0038] The Applicant has developed, as a first object of the invention, a bioabsorbable skin substitute A) for ulcers and wounds, which is obtained by subjecting a composition Al) comprising a) a cellular component comprising fibroblasts, b) a physical mixture comprising hyaluronic acid, fibrin and / or platelet-rich plasma gel; to a cryogenic procedure comprising the following steps:

[0039] - cooling at 4°C and maintaining the composition Al) at this temperature for a time comprised between 1 and 2 hours;

[0040] - freezing at -20°C and maintaining the composition Al) at this temperature for a time equal to at least 18 hours.

[0041] A second object of the invention is a skin patch C) comprising

[0042] - a layer comprising or consisting of the bioabsorbable skin substitute A);

[0043] - an outer protective layer B) comprising an adhesive layer Bl disposed directly in contact with the skin substitute A) and an outer support B2) disposed on the adhesive layer B 1.

[0044] A third object of the invention is a process for preparing the skin patch C) comprising the following stages: i) Preparing the cellular component a) comprising fibroblasts; ii) Adding the cellular component a) from the preceding stage i) to a physical mixture b') comprising hyaluronic acid, fibrinogen and / or platelet-rich plasma and obtaining a composition A2); iii) Adding a fibrinogen activator comprising or consisting of thrombin and an inorganic salt preferably selected from: Trisodium citrate, Sodium bicarbonate Sodium chloride to obtain the fibrin, and / or a platelet-rich plasma activator preferably selected from batroxobine and / or a calcium salt with an organic acid or mineral to obtain the PRP gel, to the mixture b') from stage ii) and obtain the composition Al). iv) Transferring the composition Al) from stage iii) onto the protective layer B) in contact with the adhesive layer Bl) and obtaining a patch Cl); v) Cryogenically treating the patch Cl) with a process comprising the following sequential steps: o cooling at 4 °C and maintaining the patch Cl) at this temperature for a time comprised between 1 and 2 hours; o freezing the patch Cl) at -20°C and maintaining the same at this temperature for a time of at least 18 hours; to obtain the patch C); o packaging the patch in sterile environment.

[0045] Advantages o f the invention

[0046] Overall, the invention developed by the Applicant is advantageous for the following reasons.

[0047] Firstly, the cryogenic treatment is not aimed at the cryopreservation of the cellular component, but rather at an increase in the production activity of the substances released by the cells themselves due to the thermal stress to which the cells have been subjected with the aforesaid cryogenic treatment.

[0048] This type of substances released by the cellular component are, for example, growth factors and alarmins, which favour epithelium regeneration and have anti- inflammatory activity.

[0049] It should be noted how, in Examples 1 and 6, cell viability / activity was studied at a temperature of -20°C. Cell viability is extremely reduced or eliminated at this temperature. This condition is aimed at the release of a series of signal molecules, including alarmins, capable of stimulating tissue regeneration.

[0050] On the contrary, and as shown in Example 5, at a temperature of -80°C the cells are viable. For this reason, cells are not 'activated' in the production of alarmins by means of RNA. It should be noted that the Applicant had difficulties in quantifying the RNA responsible for the production of alarmins in the experimental trials conducted on cells in suspension at -80°C. This proves that, at a very low temperature of -80°C, the release of these signal molecules is not possible. The Applicant considers that such an inactivation effect at -80°C can also be found at temperatures < 80°C.

[0051] At the same time, reducing the temperature of the cryogenic treatment is important for a second aim, namely that of reducing or eliminating the proliferation of fibroblast cultures, thereby decreasing or eliminating the problem of a possible immune response (immunogenicity) on the part of the subj ect receiving treatment with the allogenic skin substitute of the invention.

[0052] Furthermore, the invention induces a relevant clinical efficacy due to both the increase in the controlled release of the aforesaid substances produced by the cells of the cellular component following heat treatment, and associated with the presence of component b), i.e., a physical mixture comprising hyaluronic acid, fibrin and / or platelet-rich plasma gel.

[0053] Finally, the production process is lean and less complex with respect to those known in the background art. In fact, it does not require any special or time-consuming technological measures, such as those which may be related to reaching relatively low temperatures.

[0054] Other advantages will become apparent in the following detailed description of the invention.

[0055] DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 - Graph showing the relative expression of pre-freezing vs post-freezing alarmins in the study of Example 2, relative to the First trial.

[0057] Figure 2 - Graph showing the relative expression of pre-freezing vs post-freezing alarmins from the Example 2 study, relative to the Second trial.

[0058] Figure 3 - Graph showing the relative expression of pre-freezing vs post-freezing alarmins from the Example 2 study, relative to the Third trial.

[0059] Figures 4A and 4B - Skin patch C) sample of the invention, seen from above (Figure 4A) and in perspective (Figure 4B). Figures 5A-6B - Graphs showing the relative expression of biglian (Figure 5 A and 6A) and tenascin-C (Figure 5B and 6B) as a function of various temperatures [Room Temperature = RT; -20°C; -80°C],

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] Below, the Applicant describes the invention in more detail.

[0062] For the purposes of the present invention, the definition 'comprises' does not exclude the presence of further components / stages non explicitly listed after such a definition. On the contrary, the definition 'constituted by' or 'consisting of excludes the presence of such further components / stages.

[0063] For the purposes of the present invention, 'physical mixture' means a set of several substances which are not bound together by covalent or ionic bonds but at most form labile bonds of the hydrogen-bridge bond type or Van der Walls forces.

[0064] For the purposes of the present invention, the definition of platelet-rich plasma or PRP means the supernatant obtained after having subjected whole blood to centrifugation to separate the red and white blood cells.

[0065] PRP gel is defined as gel obtained from PRP placed in contact with an activating agent such as batroxobin possibly in the presence of an organic or inorganic calcium salt, more preferably calcium chloride or calcium gluconate.

[0066] The first object of the invention is the bioabsorbable skin substitute A), which is obtained by subjecting a composition Al) comprising a) a cellular component comprising fibroblasts, b) a physical mixture comprising hyaluronic acid, fibrin and / or Platelet-Rich Plasma, to a cryogenic procedure comprising the following steps:

[0067] - cooling at 4°C and maintaining the composition Al) at this temperature for a time comprised between 1 and 2 hours;

[0068] - freezing at -20°C and maintaining the composition Al) at this temperature for a time of at least 18 hours. Preferably, the cellular component a) is allogenic. In surgery, the term 'allogenic' indicates a tissue from another organism, i.e., from a donor; it is the opposite of the term 'autologous.'

[0069] Preferably, the cellular component a) is not proliferating, which means that, although it is active and / or capable of producing exogenous substances, the cellular component a) is not, at the same time, capable of proliferating or reproducing or initiating mitosis processes. The cellular component a) comprising fibroblasts is, therefore, preferably exploited as a natural source of growth factors and alarmins, protagonists of the tissue repair or regeneration process.

[0070] Preferably, the cryogenic procedure comprises the following steps:

[0071] - cooling at 4°C and maintaining the composition Al) at this temperature for 1x / i hours;

[0072] - freezing at -20°C and maintaining the composition Al) at this temperature for a time equal to 18 hours.

[0073] The Applicant considers that the cryogenic procedure, which coincides with the cryogenic treatment of the stage v) of the patch Cl) according to the process of the invention, is functional for an increase in the production of exogenous substances or cellular products by the cellular component a) comprising fibroblasts, with respect to when the cryogenic procedure is absent, in other words when the skin substitute coincides with Al).

[0074] Preferably, such cellular products are selected from the group consisting of growth factors, alarmins, interleukins, and mixtures of the foregoing.

[0075] Preferably, the alarmins (also referred to as Damage-associated molecular patterns or DAMPs) have the ability to trigger the cellular response following damage and promote the regeneration / repair of the lesion / ulcer. Their antibiotic and immuno- modulatory properties allow a faster healing of lesions, wounds or skin ulcers.

[0076] Preferably, the cellular products are selected from the group consisting of: High- Mobility Group Boxl (HMGB-1), biglycan, tenascin-C (TNC), Fibroblast Growth Factor 2 (FGF-2), IL-6, IL-8, and mixtures of the foregoing. Preferably, the bioabsorbable skin substitute A) is of allogenic type; it also preferably constitutes a carrier for the release of proteins with regenerative capacity.

[0077] It should be noted that the cellular component a) comprising fibroblasts of the skin substitute A) is obtained from the skin substitute Al) by cryogenic treatment comprising the aforesaid steps:

[0078] - cooling at 4°C and maintaining the composition Al) at this temperature for a time comprised between 1 and 2 hours;

[0079] - freezing at -20°C of the composition Al) and maintaining the same at this temperature for a time of at least 18 hours.

[0080] This cryogenic treatment, as anticipated above, coincides with stage v) of the preparation process of the patch C) which is the further subject-matter of the invention.

[0081] With respect to the intermediate Al), the skin substitute A) differs substantially in the cellular component a) which, due to freezing, contains substances

[0082] The component b) of both the skin substitute intermediate Al) consists of a physical mixture comprising hyaluronic acid, fibrin and / or Platelet-Rich Plasma gel" b).

[0083] The component b) of both the intermediate composition Al) and the skin substitute, i.e., the physical mixture comprising hyaluronic acid, fibrin and / or Platelet Rich Plasma gel" b) is obtained from a physical mixture comprising hyaluronic acid, fibrin and / or Platelet Rich Plasma, in the presence of at least one fibrinogen activator, and / or in the presence of at least one platelet-rich plasma activator, as described in stage iii) of the process subject-matter of the invention to prepare the process of the skin patch further subject-matter of the invention.

[0084] The bioabsorbable skin substitute A) is in particular suitable for the treatment of ulcers and lesions, and preferably chronic ulcers and lesions, as it allows to regenerate injured or ulcerated skin tissue.

[0085] Skin patch C)

[0086] A second object of the invention is the skin patch C) comprising

[0087] - an inner layer comprising or consisting of the bioabsorbable skin substitute A); - an outer layer B) disposed on the bioabsorbable skin substitute A) comprising an adhesive material Bl) disposed in contact with the skin substitute A) and an outer layer B2) disposed above the adhesive layer B2).

[0088] The support B2) is a sterile support, preferably transparent, more preferably it is a high-permeability polyurethane film, the adhesive layer B2 is a hypoallergenic latex- free pressure sensitive adhesive (PSA). This allows an optimal exchange of oxygen and water vapour, such as to maintain the correct degree of moisture in contact with the wound / ulcer. Furthermore, it is impermeable to liquids, bacteria and viruses and thus protects the insertion site from external contamination.

[0089] Preferably, the skin patch C) can also contain active ingredients useful for the purposes of the invention, which are not produced by the cellular component a) comprising fibroblasts; they can, for example, be contained in the adhesive layer Bl.

[0090] Use of the skin patch C)

[0091] The skin patch C) is preferably used in the regeneration of tissue, preferably epithelial or skin tissue, and in particular in the treatment of wounds, ulcers or skin lesions.

[0092] Preferably, such ulcers are of chronic type.

[0093] Preferably, the skin patch C) is applied to the injured lesion or ulcer on the side of skin substitute A) and the protective layer is removed after a period of time comprised between 15 and 23 days, preferably between 17 and 23 days, preferably between 17 and 21 days, preferably equal to 21 days.

[0094] Preparation process of the skin patch C)

[0095] A further object of the invention is the preparation process of the skin patch C) comprising the following stages: i) Preparing the cellular component a) comprising fibroblasts; ii) Adding the cellular component a) from the preceding stage i) to a physical mixture b') comprising hyaluronic acid, fibrinogen and / or Platelet-Rich Plasma and obtaining a composition A2); iii) Adding a cross-linking agent comprising or consisting of thrombin to the composition A2) from stage ii) and obtaining the composition Al); iv) Transferring the composition Al) from stage iii) onto the outer adhesive layer B) and obtaining a patch Cl); v) Cryogenically treating the patch Cl) with a process comprising the following steps: o cooling at 4 °C and maintaining the patch Cl) at this temperature for a time comprised between 1 and 2 hours; o freezing the patch Cl) at -20°C and maintaining the same at this temperature for a time of at least 18 hours.

[0096] Stage i) - Preparation of the cellular component a) comprising fibroblasts

[0097] Preferably, the stage i) of preparing the cellular component a) comprising fibroblasts comprises the following steps: i-1) arranging at least one human skin fragment obtained by means of biopsy from a skin donor; i-2) separating the epidermis from the dermis with conventional methods; i-3) treating the dermis enzymatically (or with enzymes) to isolate the fibroblast cells; i-4) culturing to confluence the fibroblast cells in a suitable culture medium, in order to obtain fibroblast cell cultures; i-5) freezing the fibroblast cell cultures obtained in the preceding stage.

[0098] Step i-1) - arranging at least one human skin fragment obtained by means of biopsy from a skin donor

[0099] Step i-1) consists of arranging at least one human skin fragment obtained by means of biopsy from a skin donor.

[0100] Preferably, a skin donor is initially selected. For example, such skin can be waste skin obtained from a weight reduction operation. The skin donor selection occurs in accordance with the European Directives on the donation, procurement, control, processing, preservation, storage and distribution of human tissues and cells (2004 / 23 / EC, 2006 / 17 / EC and subsequent updates and national transpositions).

[0101] Preferably, once the skin donor has been identified, a biopsy is performed, obtaining at least one human skin fragment. Preferably, the at least one skin fragment is preferably stored in a medium of suitable transport, then sent, by means of a qualified courier, and with controlled transport at a temperature comprised between 2°C and 8°C, to an authorised pharmaceutical laboratory for the production of cellular products.

[0102] Preferably, about 36 cm2human waste skin is collected; however, keeping the same geometry, it is possible to reduce or increase the number of flasks to be sown after the digestion step.

[0103] Step i-2) - separating the epidermis from the dermis

[0104] The separation of the epidermis from the dermis preferably occurs by means of incubation in the presence of Neutral Protease. The operating conditions are preferably as follows: 6 DMCU / cm2of tissue in 7 ml HBSS + 14pl of a IM CaCh solution, preferably for 18 h at 4°C. After enzymatic digestion with Neutral Protease, the skin fragment is washed with DPBS (Dulbecco's Phosphate Buffered Saline); it is then transferred to a Petri dish and the epidermis is separated from the dermis by means of sterile tweezers. The dermis fragment is then shredded into smaller pieces with a scalpel and transferred into a 15 mL tube. DMCU is the following compound: (3-(3,4- di chlorophenyl)- 1,1 -dimethylurea). HBSS is Hanks' Balanced Salt Solution.

[0105] Step i-3) - enzymatically treating the dermis and isolating the fibroblasts

[0106] The dermis is enzymatically treated preferably by means of collagenase. Preferably, the operating conditions are as follows: 0.3 PZU / ml in 10 ml of HBSS + 20ul of IM CaCh, preferably for 2 h at 37°C. At the end of digestion, the solution is filtered to eliminate the coarse pieces and is subjected to centrifugation to obtain a pellet of cells extracted from the tissue.

[0107] Step i-4) - culturing to confluence the fibroblasts in a suitable culture medium in order to obtain fibroblast cell cultures

[0108] Preferably, the fibroblasts are cultured and expanded, preferably expanded in a flask, in a suitable medium until confluence, preferably until 70-80% confluence.

[0109] Preferably, a suitable medium or culture medium consists of: Dulbecco's Modified Eagle Medium (DMEM), Glutamine, Sodium Pyruvate and Foetal Bovine Serum (FBS) (irradiated and certified European Directorate for the Quality of Medicines (EDQM)); the formula for the complete culture medium or Complete Medium (CM) is as follows: 500 mL DMEM, 10 mL Glutamine 200 mM, 50 mL FBS, 5 mL Sodium Pyruvate IM.

[0110] Preferably, the culture and expansion process scheme according to step i-4) is as follows:

[0111] Sowing in 6 T25 flasks

[0112] Expansion in 6 T150 flasks

[0113] Sowing in 1 cellstack of 5 levels

[0114] Performing further splits in cellstacks of 5 levels according to the required batch size while maintaining the seeding density at a value of approximately 5000 cells / cm2.

[0115] All the medium components must be sterile.

[0116] Preferably, the culture and expansion are performed under a streamline flow hood (grade A) with a grade B surrounding, while the components are added in a sterile manner in the bottle containing DMEM.

[0117] Preferably, the medium is changed every 2-4 days, checking cell growth under the microscope with each medium change.

[0118] At each split, the medium is removed from the flask, the flask is then washed with Dulbecco's Phosphate-Buffered Saline (DPBS) and recombinant trypsin is added, then the cells are seeded in the flask or cell stacks.

[0119] Step i-5) - freezing the fibroblast cell cultures obtained in the previous stage Preferably, at the end of the expansion of the previous step i-4), the fibroblast cell cultures are collected and separated into containers or vials or cryovials, preferably each vial or cryovial contains 10xl06cells.

[0120] The fibroblast cell cultures are then frozen, preferably in a suitable medium and in the presence of a cry opreserving or cryoprotectant agent. Preferably, the suitable medium is Foetal Bovine Serum (FBS or PBS); preferably, the cryopreserving agent is dimethylsulfoxide (DMSO), preferably the latter is equal to 10% with respect to the total content. Preferably, before stage ii) of adding the component a) of the preceding stage i) to a physical mixture b') comprising hyaluronic acid, fibrinogen and Platelet-Rich Plasma, the cell component a) is thawed. Preferably, for the purposes of the invention, the volume of thawed cell component a) is 1 ml. Preferably, the thawed cell component a) is placed in a 15 ml tube.

[0121] Preferably, after the thawing, a suitable medium, saline solution or PBS (Phosphate- buffered saline), preferably 9 ml PBS, is added to the cell component a). Then the cell component a) is centrifuged with the suitable medium added, then the supernatant is removed and the resulting solid (or pellet) obtained is resuspended with NaCl saline solution, preferably 200 pl NaCl.

[0122] Stage ii) - Addins the cellular component a) of the preceding stage i) to a physical mixture comprising hyaluronic acid, fibrinogen and / or Platelet-Rich Plasma

[0123] The physical mixture b') comprising hyaluronic acid, fibrinogen and / or Platelet-Rich Plasma is initially arranged.

[0124] The hyaluronic acid used in this stage is first sterilised by means of filtration through 0.2 micron filters, then aseptically added in stage ii) of the process of the invention to fibrinogen or PRP in sterile solution in an ISO 7 clean room within an ISO 5 biological safety cabin to obtain the physical mixture b').

[0125] Preferably, the amount of hyaluronic acid added in stage ii) is comprised between 0.5 and 5 mg / ml, preferably between 1 and 4 mg / ml by weight on the total volume of the physical mixture, and shows a weight average molecular weight comprised between 1000 and 3000kDa.

[0126] The cellular component suspended in suitable medium and coming from the preceding stage is then added to the physical mixture b').

[0127] Stage in) - Adding a cross-linking agent comprising or consisting o f thrombin to the primary mixture coming from stage ii) and obtaining the composition Al)

[0128] Stage iii) involves the addition of an activator to activate the fibrinogen to transform it into fibrin and / or comprising or consisting of thrombin and at least one inorganic salt, batroxobin and / or calcium salt of an organic or inorganic acid to precipitate the PRP gel to the mixture b') from stage ii), obtaining the composition Al). The organic or inorganic salt present in the aqueous thrombin solution is preferably chosen from trisodium citrate, sodium bicarbonate, sodium chloride, calcium chloride.

[0129] The thrombin is added as an aqueous, dilute solution, preferably the dilution is 1 : 40 thrombin / calcium chloride solution; this is obtained by adding 10 pl of 1000 U / ml thrombin to 390 pl of IM CaCh .

[0130] Preferably, stage iii) involves the addition of 134 pl of thrombin to the primary mixture obtained from stage ii) to initiate the polymerisation with fibrin glue formation. Preferably, during this stage iii), it is gently mixed to avoid the formation of bubbles.

[0131] Preferably, the sodium bicarbonate is at a concentration comprised between 1 and 3 mg / ml, preferably 2 mg / ml.

[0132] Preferably, the sodium chloride is at a concentration comprised between 7 and 10 mg / ml, preferably 8 and 9 mg / ml.

[0133] Preferably, the formation of skin substitute Al) is conducted at a temperature comprised between 35°C and 40°C, preferably between 36°C and 38°C, preferably equal to 37°C.

[0134] The organic or inorganic calcium salt to be added to the batroxobin to precipitate the PRP gel is selected from: calcium chloride and calcium gluconate.

[0135] This combination of hyaluronic acid fibrin and / or PRP gel obtained in stage iii) and contained in the skin substitute A. which is the subject-matter of the invention and in turn also contained in the patch C., allows to obtain a safe and stable product. In fact, the mixture of hyaluronic acid fibrin and / or PRP gel associates the viscoelastic, protective and anti-inflammatory properties typical of hyaluronic acid with those of a fibrin-based matrix and / or PRP gel to generate a unique biocompatible environment which stimulates the skin's physiological regenerative processes.

[0136] Stage iv) - Transferrins the composition Al) from stage iii) onto the outer adhesive layer B) and obtaining a patch Cl)

[0137] Preferably, a protective layer B) comprising a transparent polyurethane film-based support B 1 is initially arranged, to which an adhesive layer B2 comprising a pressure- sensitive adhesive (PSA) which is hypoallergenic and therefore latex-free is applied. Preferably, such a protective layer B) comprising both the support B2 and the adhesive layer B 1 is commercially available. Preferably, the size of the protective adhesive layer B) is 10x12 cm.

[0138] Preferably, the packaging of the protective adhesive layer B) is opened in the streamline flow hood, then the protective adhesive layer B) is positioned in a capsule or Petri dish, preferably rectangular and of adequate size.

[0139] The composition Al) is preferably transferred immediately, on the surface of the outer protective layer B) from the adhesive layer Bl, disposed inside a Petri dish, to obtain a patch Cl). Preferably, the Petri dish is covered with a suitable lid.

[0140] Preferably, the patch Cl) is left at room temperature (RT) for a time comprised between 15 and 25 minutes, preferably 20 minutes. Room temperature is intended as a temperature comprised between 18°C and 25°C, preferably not exceeding 25°C.

[0141] Stage v) - Cryogenic treatment of the patch Cl).

[0142] Then, after the required time has elapsed, a suitable freezing solution is prepared and disposed over the patch Cl), preferably covering the latter with at least one layer (veil) of freezing solution.

[0143] Preferably, the freezing solution consists of 90% NaCl saline solution and 10% dimethylsulfoxide (DMSO). Preferably, the freezing solution to be disposed on the patch Cl) is equal to 1 ml.

[0144] Preferably, the cryogenic treatment of the patch Cl) occurs in a cryogenic process comprising (or consisting of) the following sequential sub-steps: o cooling at 4 °C and maintaining the patch Cl) at this temperature for a time comprised between 1 and 2 hours; o freezing at -20°C of the patch Cl) and maintaining the same at this temperature for a time equal to at least 18 hours and until final use.

[0145] Stage vi) packaging the patch in a sterile atmosphere

[0146] The patch Cl) is transferred, from inside the Petri dish, to inside a suitable container, preferably inside an aluminium bag; then, this suitable container is sealed, preferably heat-sealed, at the end and labelled. EXAMPLES

[0147] The Applicant provides the examples hereinafter for merely illustrative and non- limiting purposes.

[0148] Example 1 - In vitro study of cell viability.

[0149] The cell viability of the fibroblasts inserted within the skin patch C) of the invention was assessed, after freezing. The reagent Alamar Blue and the corresponding colorimetric assay was used, which provides an indication of the metabolic activity of the cells by calculating the percentage reduction of the Alamar Blue reagent.

[0150] 1.1 First trial (27 / 4 / 2021)

[0151] The freezing included a passage at 4°C for I’A hours and one at -20°C for 18 hours.

[0152] At each time point, the metabolic activity of the fibroblasts was assessed, which correlates with cell viability.

[0153] Table 1

[0154] The data in Table 1 highlighted a residual metabolic activity of 4% at -20°C. The results therefore indicate that freezing leads to high fibroblast mortality. This condition is essential for the cells to release a whole series of signal molecules, including alarmins, capable of stimulating tissue regeneration.

[0155] 1.2 Second trial (24 / 11 / 2021)

[0156] In this trial, the metabolic activity of three different batches of fibroblasts (FB batch 1, FB batch 2 and FB batch 3) was analysed after the scaffold was left at -20°C for 18 hours, 7 days, 14 days and 28 days.

[0157] Table 2

[0158] The data in Table 2 highlighted a residual metabolic activity at -20°C after 18h: 4% for FB batch 3, confirming the data from the first trial; 11% for FB batch 1 and 9% FB batch 2.

[0159] After 7 days at -20°C: the activity for FB batch 2 is negative, while for FB batch 1 and FB batch 3 it seems to increase slightly to 17% and 11%, respectively.

[0160] After 14 days at -20°C, the metabolic activity of: FB batch 1 goes to zero; FB batch 2 has a percentage of 4% and FB batch 3 of 12%.

[0161] After 28 days at -20°C, the metabolic activity of: FB batch 1 becomes negative, FB batch 2 passes to 7% and FB batch 3 decreases to 8%.

[0162] Considering the results obtained, we conclude that a residual metabolic activity below 12% is not related to cell viability, but to the presence of enzymes among the cell debris which are still capable of reducing the Alamar Blue reagent used for the assay.

[0163] Thus, it is confirmed that freezing at -20°C leads to a high fibroblast mortality already after 18 hours. This condition is essential for the cells to release a whole series of signal molecules, including alarmins, capable of stimulating tissue regeneration.

[0164] Example 2 - In vitro evaluation of alarmin expression levels following the freezing process.

[0165] The analyses focused on the production of alarmins by the fibroblasts inserted in the scaffold.

[0166] RT-PCR (Real Time-PCR) assays were performed to assess the expression levels of the alarmins HMBG1, Biglycan and Tenascin-C, fibroblast growth factor (FGF2) and interleukins (IL-6 and IL-8).

[0167] Genes observed in Real Time:

[0168] - High-Mobility Group Boxl (HMGB1) is a non-histone nuclear protein. In physiological situations, it is ubiquitously present and regulates DNA transcription, replication and repair. In stress situations, it is secreted in hyper-acetylated form and supports tissue regeneration by stimulating cell activation, differentiation and migration (fibroblasts, monocytes / macrophages, dendritic cells and endothelial cells).

[0169] - BIGLYCAN (BGN) is a proteoglycan present on the cell membrane, where it is capable of interacting with various extracellular molecules (TNF-alpha, TGF-beta, cytokines, growth factors, etc.). Following stress, the cell increases production of biglycan, which is released extracellularly. In its soluble form, it acts as an alarmin by interacting with the cells of the immune system (macrophages and neutrophils) and activating the immune response.

[0170] - TENASCIN-C (TNC) is an extracellular matrix glycoprotein with regulatory action on inflammation. Following tissue damage or cellular stress, its expression is up- regulated in the first 24 hours to allow the inflammatory state to resolve.

[0171] - Fibroblast Growth Factor 2 (FGF2) is the growth factor expressed by fibroblasts and will be used as a cell marker.

[0172] - IL-6 and IL-8 are two cytokines which mediate inflammation. In particular, IL-8 has pro-inflammatory activity.

[0173] The analyses were performed by comparing the relative expression

[0174] - pre-freezing (immediately after polymerization) and

[0175] - post-freezing (after 18h at -20°C).

[0176] 2.1 First trial (31 / 3 / 2021 and 14 / 4 / 2021)

[0177] Figure 1 shows that, for this first trial, the dermal-derived fibroblasts express the studied alarmins. The heat shock, performed by moving to 4°C and subsequent freezing at -20°C, led to a change in the expression of the alarmins considered, with the exception of HMGB1. It is further observed that the freezing ramp used favours the increased expression of Biglycan and Tenascin-C and decreased inflammatory cytokines IL-6 and IL-8.

[0178] Since our interest is in alarmins, only Biglycan, Tenascin C, HMGB1 and the growth factor FGF2 as a fibroblastoid marker were analysed in the next two trials (Second and Third). 2.2 Second trial (08 / 06 / 2021)

[0179] As shown in Figure 2, the results confirm the previous analyses: the freezing process leads to an increase in the expression levels of the alarmins Biglycan and Tenascin C, proving to be an important step for the presence of pro-regenerative factors in the final product.

[0180] 2.3 Third trial (20 / 10 / 2022)

[0181] As can be seen in Figure 3, the data again confirm the results of the other two trials: the freezing process leads to an increase in the expression of the alarmins Biglycan and Tenascin C. In addition, an increase in HMGB1 alarmin levels is also noted.

[0182] Example 3 - In vitro evaluation of alarmin production and release following the freezing process.

[0183] We first proceeded with the total quantification of the proteins released by the fibroblasts inserted in the scaffold after the freezing process.

[0184] The samples taken into consideration are

[0185] - the scaffold or skin patch with fibroblasts inside (called 'gel+cells') and

[0186] - the scaffold or skin patch without cells (referred to as 'gel only' or 'white'), which was used as a blank (inside the scaffold there is fibrinogen / fibrin, HA and thrombin), after the freezing procedure at -20°C and 18h.

[0187] The BCA (Micro Bicinchoninic Acid (BCA) protein assay) data are summarised in Table 2 below.

[0188] Table 2 Subsequently, using Elisa assays, we wanted to verify and quantify the presence of alarmins produced and released by the fibroblasts inserted inside the scaffold following freezing.

[0189] The targets studied are HMBG1, Biglycan, Tenascin-C and fibroblast growth factor (FGF2).

[0190] Table 3 summarises the data in pg / ml obtained during the two trials performed. The samples taken into account are the scaffold with fibroblasts inside ('gel+cells') and the scaffold without cells ('gel only'), which was used as a negative control (there must be no alarmins in the gel only), after the controlled freezing ramp and 18h at -20°C.

[0191] Table 3 [ND = Not Detected]

[0192] The analyses show that the fibroblasts release proteins inside the scaffold. The skin patch product of the invention will therefore be rich in these regenerative factors useful for healing ulcers.

[0193] The Applicant notes that, in the presence of 'gel only,' it is possible to observe a certain value, for example, of FGF2 because a cross-reactivity phenomenon or the formation of an ^specific binding of the secondary antibodies of the ELISA assay to the proteins present in the gel only could occur.

[0194] Example 4 - In vitro evaluation of the pre-freezing and post-freezing alarmin quantity.

[0195] Following the results obtained, we wanted to understand whether the freezing process (Ih 30 min at 4°C and then at -20°C) could stimulate an increase in the production of the alarmins Biglycan, Tenascin C and HMGB1. Two gels containing the cells ('gel+cells') were set up. Following polymerisation, one was used to extract the proteins before freezing (PRE-FREEZING sample) while the other was first frozen and then used for the protein extraction (POST-FREEZING sample). Total protein quantification by means of BCA:

[0196] Table 4

[0197] The values obtained from the ELISA assays performed are summarised in table 5 below (pg / ml).

[0198] Table 5 Comparing the pre-freezing sample with the post-freezing one shows a 40% increase in HMGB1 and 80% increase in Biglycan, but not in Tenascin C, which remains at the same order of magnitude. The Applicant notes out that cryogenic treatment is indispensable for an enrichment of pro-regenerative factors such as alarmins.

[0199] The Applicant points out that, for the example 4 in question, there is an increase in HMGB 1 but not in TNC with respect to the trials in example 2. This is due to the type of assay which was performed. In example 2, the expression of messenger RNA in the cell which will go to encode certain proteins is assessed, while in example 4, the proteins extracted from the gel+cell are quantified. Example 5 - Evaluation of alarmin expression at different temperatures.

[0200] A set of experiments were carried out to identify the freezing process capable of causing an increase in the levels of alarmins produced by the fibroblasts seeded within the hyaluronic acid ('HA') and fibrin scaffold.

[0201] The cell and gel samples were all prepared and treated in the same manner. The RNA was extracted before freezing, and then at temperatures of -20°C and -80°C. The RNA thus obtained from the samples allowed to proceed with reverse transcription and then RT-PCR (Real Time-PCR) assays to evaluate the expression levels of the alarmins biglycan and tenascin-C.

[0202] As can be seen in Figure 5 A, the expression of biglycan increases after freezing at - 20°C, then decreases at -80°C. Similarly, as can be seen from Figure 5B, the expression of tenascin-C also increases after freezing at -20°C while it decreases at -80°C.

[0203] The thermal stress to which the cells were subjected showed that keeping the samples at -20°C induced an increase in the RNA expression of the alarmins, whereas at -80°C their expression was similar to that obtained at room temperature.

[0204] These results were confirmed by a second set of experiments performed using the same procedures (see Figures 6A and 6B).

[0205] Example 6 - Stability of the fibroblast cell component.

[0206] A stability assay of dermal fibroblasts was performed to demonstrate that their viability remains high over time, and that it is only during the scaffold preparation process, when the HA-Fb ('hyaluronic acid-fibrin') and cell mixture is transferred to -20°C, that cell mortality occurs with the advantageous outcome of stimulating the release of alarmins by the fibroblasts. It should be noted that the cells, before being mixed with HA-Fb, are thawed in saline solution, centrifuged and the pellet resuspended in 0.2 ml of saline solution.

[0207] The dermal fibroblasts produced were frozen in cryovials, in a suitable freezing solution (Saline solution and 10% DMSO) and, at the time of freezing, the viability was 96%. The cryovials prepared at time zero for stability were kept in liquid nitrogen until the various thawing time points.

[0208] In Table 6, cell stability data in saline solution and 10% DMSO are shown.

[0209] Table 6

[0210] The data obtained show that the cells, after thawing, before being placed in the HA-Fb scaffold, have a high viability of 85% or more, and it is the freezing process of the final product at -20°C which induces a programmed fibroblast mortality, causing the release of alarmins.

[0211] This finding was confirmed by an assay of fibroblast metabolic activity which correlates with cell viability (table 7).

[0212] Table 7

[0213] The results therefore indicate that the freezing leads to a high fibroblast mortality. This condition is essential for the cells to release a whole series of signal molecules, the alarmins, capable of stimulating tissue regeneration.

Claims

CLAIMS1. Bioabsorbable skin substitute A) for ulcers and wounds, obtained by subj ecting a composition Al) comprising: a) a cellular component comprising fibroblasts, b) a physical mixture comprising hyaluronic acid, fibrin and / or Platelet Rich Plasma gel, to a cryogenic procedure comprising the following sequential steps: cooling at 4 °C and maintaining the composition Al) at this temperature for a time comprised between 1 and 2 hours; freezing the composition Al) at -20 °C and maintaining the same at this temperature for a time of at least 18 hours.

2. Skin substitute according to claim 1, wherein the cellular component a) is allogenic.

3. Skin patch C), comprising: a layer comprising or consisting of the bioabsorbable skin substitute A) according to claim 1 or 2; an outer protective layer B) disposed on the skin substitute A), comprising an adhesive layer Bl) disposed in direct contact with the skin substitute A), and an outer support B2) disposed on the adhesive layer Bl).

4. Skin patch, wherein the adhesive layer B) is a polyurethane film comprising a non-allergenic, latex-free pressure-sensitive adhesive (PSA).

5. Process for preparing the skin patch C) according to claim 3 or 4, comprising the following stages: i) preparing the cellular component a) comprising fibroblasts; ii) adding the cellular component a) from the preceding stage i) to a physical mixture b’) comprising hyaluronic acid, fibrinogen and / or Platelet Rich Plasma and obtaining a mixture A2); iii) adding a fibrinogen and / or rich plasma gel activator to the composition A2 from stage ii) and obtaining the composition Al according to claim 1, said activator comprising or consisting of thrombin and at least an organic or inorganic salt of alkali or alkaline-earth metal, in order to precipitate thefibrin, and / or a calcium salt with and organic or inorganic acid, in order to precipitate the platelet gel; iv) transferring the composition Al) from stage iii) on the outer layer B) in contact with the adhesive layer Bl) and obtaining a patch Cl); v) cryogenically treating the patch Cl) with a process comprising the following steps: o cooling at 4 °C and maintaining the patch Cl) at this temperature for a time comprised between 1 and 2 hours; o freezing the patch Cl) at -20 °C and maintaining the same at this temperature for a time of at least 18 hours; vi) packaging the patch in sterile environment.

6. Process according to claim 5, wherein stage i) comprises the following steps: i-1) providing at least one human skin fragment obtained through biopsy from a skin donor, i-2) separating epidermis from dermis; i-3) enzymatically treating the dermis, in order to isolate fibroblast cells; i-4) culturing to confluence the fibroblast cells in a suitable culture medium, in order to obtain fibroblast cell cultures; i-5) freezing the fibroblast cell cultures obtained in the preceding stage.

7. Process according to claim 5 or 6, wherein before stage ii) the cell component a) is thawed.

8. Process according to any one of claims from 5 to 7, wherein stage v) is carried out in the presence of a cryopreserving agent, preferably dimethyl sulfoxide.

9. Patch according to claim 3 or 4, for use in treating wounds, ulcers or skin injuries.

10. Patch for use according to claim 9, wherein said ulcers are of chronic type.

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