IT202100017411A1 - Method of decellularization of tissues and organs - Google Patents

Description

DESCRIPTION

Attached to a patent application for INDUSTRIAL INVENTION having the title

?Method of decellularization of tissues and organs?

FIELD OF THE INVENTION

The present invention relates to a decellularization method of tissues, in particular the dermis and skin, and organs intended for clinical use.

BACKGROUND OF THE INVENTION

Connective tissue transplant? an operation that often ? capable of saving a person's life.

A typical example? given by skin transplantation, which is used in extensive third and second degree burns of the body. People who suffer burns in most of the body risk their lives as the lack of skin lining causes loss of fluids and proteins, drastically lowers body temperature and directly exposes the body to physical and microbiological agents.

When are the burns not very extensive? It is possible to perform self-transplants of skin, taking the skin from the non-burned parts of the same person and implanting it on the burns.

In autologous transplants, the tissue necessary for the transplant is taken directly from the subject who will receive it. the transplanted tissue.

In these types of transplant there are no complications due to immune reactions and rejection, as the transplanted tissue is recognized by the body and therefore is not attacked by the immune defences.

However, in some cases autologous transplants are not feasible. In fact, for example, in children and in very extensive burns, there is not enough intact skin to carry out a self-transplant.

In these cases homologous or heterological transplants are used, that is to say transplants in which the donor is of the same species of the receiver but not ? the recipient himself or in which the donor ? of a different species from that of the recipient.

These transplants, however, are subject to serious post-operative complications due to immunological reactions and rejection of the transplanted tissue.

The rejection? that complex of biological reactions on the basis of which the organism tends to reject the transplanted tissue recognizing it as foreign.

To obviate this serious drawback, methods have been developed aimed at achieving immune tolerance, i.e. biological acceptance, by the receiving organism of the foreign tissue that is being transferred to it. been grafted.

A first type of method provides for the administration of large doses of immunosuppressive drugs, for example "cyclosporine", to the subject who has undergone the transplant.

This methodology, if on the one hand decreases the probability? of rejection, on the other hand exposes the transplanted subject, however already? weakened by the operation undergone, to the probable contraction of infections which cannot be autonomously contrasted by the transplanted body since the entire immune system is depressed by the administered drugs.

Another type of method developed to try to avoid the rejection of the transplanted tissue involves treating the tissue taken from the donor before grafting it into the recipient subject.

This methodology? directed on the one hand to stabilize the protein and collagen structures of the extracellular structure of the tissue taken and, on the other hand, to mask or eliminate the antigenic agents present in the tissue taken. Numerous scientific works have demonstrated the possibility to treat the tissue in the way described above to make it ?acellular? and thus avoid immune reactions or rejection.

A known example of such work? given by a method of obtaining an acellular dermal implant obtained from human cadaver skin.

This method involves eliminating the cells responsible for the immunological reactions from the tissue by using detergent substances, such as polyoxyethylene, sodium deoxycholate and the like.

Alternatively, instead of detergent substances, the use of enzymes or particular salts is described.

Subsequently, this method involves placing the treated tissue in a cryoprotective liquid and dehydrating it. Freezing allows you to keep the extracellular matrix of the tissue intact.

The fabric what? obtained is thawed and rehydrated before transplantation and, once transplanted, it is rapidly revascularized by the blood of the transplanted person.

The Applicant of the present patent application has already developed a method for de-cellularizing tissues and/or organs object of the patent application WO2009050571. This method? particularly useful for treating the dermis in the case of homologous transplants and ? based on a non-invasive and non-toxic technology for the tissue, which employs a trypsin-based enzymatic solution able to phagocytose at least in part fibroblasts, macrophages, mast cells, and other cells responsible for immune reactions and rejection in transplants.

The decellularised dermis obtained with this method? successfully used in various clinical-transplantation fields since 2009 without ever leading to adverse reactions of any kind on recipient patients, both immediately and in the various follow-ups over time.

The dermis treated with this method is taken from the anatomical area of the trunk of multi-organ and/or multi-tissue donors, an area physiologically less rich in skin appendages, while the known method is not used to treat dermis taken from other anatomical areas such as, for example, the upper or lower limbs of the donor.

The known method has limitations in the treatment of dermis originating from areas physiologically rich in skin appendages, such as for example the upper or lower limbs, since it does not allow to achieve a decellularization sufficient to avoid or significantly decrease the rejection reactions.

On the other hand, the use of dermis from the donor trunk poses limitations:

? both in terms of quantity? total (in cm<2>) of decellularised dermis produced and therefore potentially distributable for clinical use to the various requesting centers (from the donor trunk area a maximum of 400-500 cm<2 > of dermis can be produced -cellularised);

? both in terms of lot size (in cm<2>) and ? it is possible to take part of the trunk area (it is possible to produce single batches of maximum dimensions 17x8 cm<2>).

There are numerous and growing clinical requests for homologous decellularised dermis, especially of large dimensions (even greater than the maximum dimensions, 17x8 cm<2>, obtainable from the trunk) for various uses in plastic surgery for the treatment of incisional hernias and extensive substance of the abdominal wall and in the breast field for the reconstruction of the breast? One step?, that is? in a single operation, in patients undergoing mastectomy.

The Applicant has therefore developed a new decellularization method which allows to maintain a high quality standard of the final product, but at the same time allows a satisfactory decellularization of tissues, such as the dermis of the upper and lower limbs, which are particularly rich in skin appendages .

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a decellularization method of an isolated tissue and/or organ intended for clinical use, i.e. to transplantation in a patient who needs it, wherein the method comprises a step of immersion of a tissue and/or organ in an aqueous solution comprising: at least one antiseptic agent, at least one polymer containing at least one positively charged quaternary ammonium and at least one surfactant . Preferably, the solution also comprises at least one stabilizing and emulsifying agent and/or at least one chelating agent.

The tissue and/or organ are kept in the aqueous solution for at least 1 hour. In a second aspect, the invention relates to the aqueous solution comprising the ingredients listed above and its use for the decellularization of tissues and/or organs.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the results of the histological analysis by H&E staining of the de-epidermal dermis (Ded) as such, subjected to a known decellularization method and to the method of the invention;

Fig. 2 shows the results of the histological analysis of the de-epidermal dermis (Ded) de-cellularised with the method of the invention by staining with Masson's trichrome;

Fig. 3 shows the results of the histological analysis of the de-epidermal dermis (Ded) de-cellularised with the method of the invention by Weigert staining;

Fig.4 shows the results of the analysis of vitality? cell of the Ded partially decellularized with the known drugs and of the Ded decellularized with the method of the invention;

Fig.5 shows the results of the cytotoxicity analysis? of Ded decellularized with the method of the invention;

Fig. 6 shows the capacity? of repopulation of the decellularized Ded with the method of the invention;

Fig. 7 shows the capacity? maximum load of Ded DEC maintained for 2 years in the solution of the invention compared to cryopreserved Derma DEC;

Fig. 8 shows the tensile strength of Ded DEC maintained for 2 years in the solution of the invention compared to cryopreserved Derma DEC;

Fig.9 shows the Young elastic modulus of Ded DEC maintained for 2 years in the solution of the invention compared to cryopreserved Derma DEC;

Fig. 10 shows the stiffness? of Ded DEC maintained 2 years in the solution of the invention compared to cryopreserved Derma DEC.

DETAILED DESCRIPTION OF THE INVENTION

The method for de-cellularizing a tissue and/or organ according to the invention comprises:

a) immersing an isolated tissue and/or organ in an aqueous solution comprising: at least one polymer containing at least one positively charged quaternary ammonium, at least one antiseptic agent and at least one surfactant; c) leaving the tissue and/or organ immersed in the aqueous solution for at least 1 hour.

The de-cellularization method ? a decellularization method at room temperature. By room temperature, in the context of the present invention, we mean a temperature higher than the freezing point of the aqueous solution, preferably between 5°C and 35°C, plus? preferably between 10?C and 30?C, even more? preferably between 15°C and 25°C or between 18°C and 25°C. The ideal maintenance temperature of the container comprising the aqueous solution and the tissue and/or organ? between 18°C and 23°C, preferably between 20°C and 23°C.

The fabric that can be de-cellularized with the method of the invention ? chosen between:

? Skin: made up of the epidermal layer containing all the cellular components that make it up and a thin layer of underlying dermis (dermo-epidermal tissue);

? De-Epidermised Dermis (Ded): Consists of dermis devoid of the overlying epidermal layer.

Preferably, the fabric ? de-epidermal dermis (Ded) homologous or heterologous, that is? coming from an individual different from the recipient of the transplant, of the same species (homologous) or of a different species (heterologous).

Preferably, the Ded ? taken from the trunk or lower or upper limbs of the human or animal donor. Pi? preferably, the Ded ? taken from the lower or upper limbs of a human or animal donor.

In one embodiment, the method of the invention is applied to a tissue or organ that ? been subjected to a decellularization method known in the field, such as for example the one described in WO2009050571. In the case of Ded taken from the trunk or from the lower or upper limbs of the donor, the known techniques, in some cases, do not allow to reach a satisfactory level of de-cellularization, ie? a level of removal of cellular annexes that does not give rise to rejection reactions in the recipient. In this case, especially for the DED taken from the upper or lower limbs of a donor, the method of the invention allows to achieve a substantial cellular removal, sufficient not to create rejection reactions in the recipient. This occurs both in the case of direct treatment of Ded with the method of the present invention, and in the case of pre-treatment of Ded with a known de-cellularization technique, such as for example the technique of WO2009050571, and subsequent treatment with the method of the invention.

The organ decellullarized with the method of the invention ? any organ taken from the human or animal body.

In the aqueous solution, the polymer containing at least one positively charged quaternary ammonium ? a polyquaternium. Polyquaternium ? the name INCI (International Nomenclature for Cosmetic Ingredients) used to describe numerous polycationic polymers belonging to different chemical classes and having in common the presence of at least one quaternary ammonium, preferably more? quaternary ammonium groups. Within the definition ?polyquaternium? INCI has approved at least 40 different polymers which are indicated with a progressive number assigned on the basis of the order of registration and not on the basis of their chemical nature.

For example, the polyquaternium-2 ? poly[bis(2-chloroethyl) ether-alt-1,3-bis[3-(dimethylamino)propyl]urea]; polyquaternium-6 ? poly(diallyldimethylammonium chloride); polyquaternium-48 ? a polymeric quaternary ammonium salt formed from methacryloyl ethyl betaine, 2-hydroxyethyl methacrylate and methacryloyl ethyl trimethyl ammonium chloride.

Therefore, polyquaterniums are polymers of different chemical nature containing at least one quaternary ammonium which can be salified for example with a chloride, a sulphide, etc.

Preferably, the polyquaternium employed in the present aqueous solution is chosen from any of the polyquaternium from polyquaternium-1 to polyquaternium-48, cio? any of polyquaterniums 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48, and combinations thereof. In one embodiment, the polyquaternium employed in the present solution is a mixture of two or more? polyquaternium selected from polyquaternium 1 to 48.

The correspondence between the numbering of the polyquaternium and the respective chemical names? note and ? therefore not fully reported here.

The polymer containing at least one positively charged quaternary ammonium ? present in the solution in quantity? between 0.001% and 0.010% by weight, preferably between 0.003% and 0.006% by weight.

The least one antiseptic agent ? selected from: polyhexaalkylenebiguanidine, preferably polyhexamethylenebiguanidine or polyhexaethylenebiguanidine, glycerol and a combination thereof.

The least one antiseptic agent ? present in the solution in quantity? between 0.0001% and 1%, preferably between 0.0001% and 0.8% by weight.

In the event that the? antiseptic agent? glycerol, it ? preferably included in the solution in quantity? from 0.1% to 1% by weight, preferably from 0.4% to 0.8% by weight.

Alternatively, the at least one antiseptic agent is present in the solution in quantity? between 0.0001% and 0.005% by weight, preferably between 0.00015% and 0.003% by weight.

The at least one surfactant ? preferably a non-ionic, hydrophilic surfactant. Preferably the surfactant ? a poloxamer. Poloxamers are a class of polymeric nonionic surfactants with a three-block structure comprising a central hydrophobic polyoxypropylene or propylene glycol chain side-linked to two hydrophilic polyoxyethylene or polyethylene glycol (PEG) chains. An example of poloxamer that can? be used in the solution of the invention ? the poloxamer 407 which has a PEG chain length of about 100 units? repetitive and a propylene glycol block length of approximately 56 units? repetitive. The at least one surfactant ? present in the aqueous solution in quantity? between 0.05% and 0.5% by weight, preferably between 0.1% and 0.3% by weight.

Preferably, the aqueous solution further comprises at least one ingredient selected from: a stabilizing and emulsifying agent and/or a chelating agent.

The chelating agent ? preferably selected from: EDTA and/or its salts, preferably the sodium salt of EDTA, and polyamine acetylacetonate.

The quantity? of chelating agent present in the solution? between 0.005% and 0.5% by weight, preferably between 0.01% and 0.1% by weight.

The at least one stabilizing and emulsifying agent, if any, is selected from hydroxyalkylcellulose, preferably hydroxymethylcellulose.

The at least one stabilizing and emulsifying agent ? present in the solution in the quantity? necessary (to taste, just enough) to obtain an adequate emulsion of the ingredients.

In one embodiment, the aqueous solution comprises: polyhexaalkylenebiguanidine, preferably polyhexamethylenebiguanidine, or glycerol, at least one polyquaternium from 1 to 48 or a mixture of polyquaternium and a poloxamer or a mixture of poloxamers.

In one embodiment, the aqueous solution comprises: polyhexaalkylenebiguanidine, preferably polyhexamethylenebiguanidine in an amount between 0.0001% and 0.005% by weight, preferably between 0.00015% and 0.003% by weight, at least one polyquaternium from 1 to 48 or a mixture of polyquaternium in quantity? between 0.001% and 0.010% by weight, preferably between 0.003% and 0.006% by weight and at least one surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount between 0.05% and 0.5% by weight, preferably between 0.1% and 0.3% by weight.

In one embodiment, the aqueous solution comprises glycerol in an amount from 0.1% to 1% by weight, preferably from 0.4% to 0.8% by weight, at least one polyquaternium from 1 to 48 or a mixture of polyquaternium in quantity between 0.001% and 0.010% by weight, preferably between 0.003% and 0.006% by weight and at least one surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount between 0.05% and 0.5% by weight, preferably between 0.1% and 0.3% by weight.

In one embodiment, the aqueous solution comprises: polyhexaalkylenebiguanidine, preferably polyhexamethylenebiguanidine, or glycerol, at least one polyquaternium from 1 to 48 or a mixture of polyquaternium, at least one surfactant, preferably a poloxamer or a mixture of poloxamers, hydroxyalkylcellulose, preferably hydroxymethylcellulose, and/or EDTA.

In a preferred embodiment, the aqueous storage solution comprises:

? polyhexaalkylenebiguanidine, preferably, polyhexamethylenebiguanidine, in an amount between 0.0001% and 0.005% by weight, preferably between 0.00015% and 0.003% by weight, and/or glycerol in an amount from 0.1% to 1% by weight, preferably from 0.4% to 0.8% by weight;

? at least a polyquaternium from 1 to 48 or a mixture of polyquaternium, the cio? any of the polyquaternium from 1 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 48 and combinations thereof, in quantity? between 0.001% and 0.010% by weight, preferably between 0.003% and 0.006% by weight;

? a non-ionic hydrophilic surfactant, preferably a poloxamer or a mixture of poloxamers, in quantity? between 0.05% and 0.5% by weight, preferably between 0.1% and 0.3% by weight;

? preferably, hydroxyalkylcellulose, more? preferably hydroxymethylcellulose, in quantity? just enough (to taste) to obtain an adequate emulsion of the ingredients; and/or

? preferably, EDTA and/or a salt thereof, plus? preferably the sodium salt of EDTA, in a quantity? between 0.005% and 0.5% by weight, preferably between 0.01% and 0.1% by weight.

In step c) of the decellularization method, the tissue and/or organ is left immersed in the aqueous solution preferably for at least 1 hour, preferably for at least 2 hours, plus? preferably for at least 3 hours, more? preferably for at least 10 days.

Advantageously, the tissue and/or organ ? left in the solution for between 1 hour and 30 days, preferably between 2 hours and 20 days, preferably between 10 and 20 days or between 10 and 25 days.

The applicant has verified, through the experiments included in the present patent application, that the Ded, preferably taken from the upper or lower limbs of a donor, therefore more? rich in skin appendages, already? after a few days it shows effective removal of the residual epidermal layer and that after 20 days the fibroblasts present in the underlying dermal matrix are sufficiently removed.

The purpose of the de-cellularization treatment of a tissue and/or organ? that of obtaining a tissue and/or organ free, at least in part, of fibroblasts, macrophages, mast cells, and other cells responsible for immune reactions and rejection in transplants.

Is the method of the invention ? proved to be very efficient in the decellularization of a tissue very rich in skin appendages such as Ded of the upper and lower limbs, thus proving to be better than the known method described in WO2009050571 for this type of tissue.

Vitality? tissue cell measured after immersion in the aqueous solution for at least 1 hour ? between 5% and 20%, preferably between 8% and 15%.

Note that in order to define a cellularly viable tissue the value of vitality? must be greater than 50%.

At the same time, ? It has been demonstrated that the aqueous solution guarantees perfect biological conservation of the extracellular matrix of the tissue, i.e. the extracellular matrix remains intact in its protein structure, in its own collagen matrix and in all this. that they are not cells responsible for the rejection reactions.

The integrity of the extracellular matrix? both biological (in the sense mentioned above) and mechanical, in the sense that the properties? mechanics of the fabric, such as for example elasticity, tensile strength and the like, remain almost unchanged compared to a fabric that has not undergone any treatment.

? it has also been experimentally noted that the tissue treated with the aqueous solution guarantees perfect growth of technocytes, thus allowing excellent cellular regrowth (with the recipient's own cells) once the transplant has been performed.

The fabric what? treaty ? also an excellent support for stem cells.

With particular reference to Ded, the method of the invention has numerous advantages.

In particular, the possibility to treat a greater overall quantity of Ded and obtain an effective decellularization, as the starting tissue can? also be taken from the upper and/or lower limbs of the donors and effectively decellularised despite being very rich in skin appendages. In particular, with the method of the invention it is possible to treat quantities equal to 1000-3000 cm<2 > of skin taken from the upper or lower limbs of the donor, while with the method of WO2009050571 only 400-500 cm<2 > of skin obtained from the donor torso could be decellularised, an area less rich in annexes cell phones but, at the same time, with less availability? of removable tissue.

Other advantage? the possibility? to produce single de-cellularized patches of larger dimensions? high (minimum 10x5 maximum 100x8 cm<2 >vs minimum 3x2 maximum 17x8 cm<2 >obtainable with the method known from WO2009050571) thus overcoming the problem of the anatomical limit of the sampling area (upper/lower limbs vs trunk).

From these two advantages derive important clinical effects especially in the two main fields of application of the de-cellularized homologous Ded and in particular:

? in the field of emergency surgery where it becomes possible to use a single patch of de-cellularized Ded of the appropriate size without having to suture multiple? patches between them. The suture is currently used, in the absence of a single patch of adequate size, to obtain a network of patches to be used in the reconstruction of the entire abdominal wall affected by incisional hernia. The network obtained from? union of pi? patch has a lower mechanical resistance to traction and more? scarring;

? in the field of plastic and breast surgery, in particular for the application of the ?one step? technique which provides for a single breast reconstruction surgery in patients undergoing mono or bilateral mastectomy following tumours. This surgical technique normally requires more? tissue patch for a single patient, at least 13x7 cm in size<2>. Thanks to the possibility to prepare patches of decellularized Ded much more? large, with the method of the present invention, this surgical procedure can? be applied more easily, with important clinical repercussions on the patients treated, in terms of less post-operative pain, reduced hospital stay times and the possibility of to avoid subsequent interventions to complete the reconstruction.

Finally, the invention also relates to an aqueous solution as described above and its use to de-cellularize a tissue and/or an organ as described above, possibly already subjected to a decellularization method. In the latter case, the method of the invention also comprises a pre-treatment of the tissue and/or organ which leads to a partial de-cellularization of the same.

In particular, the method of the invention comprises a pre-treatment, before step a), as follows:

a1) immersion of the tissue and/or organ in an enzymatic solution capable of engulfing at least in part fibroblasts, macrophages, mast cells, and other cells responsible for immune reactions and rejection in homologous and heterologous transplants;

a2) irradiation of the tissue or organ cos? treated with ionizing electromagnetic radiation, preferably having a frequency between 10<19 > and 10<22 >Hz.

The enzyme solution? preferably a trypsin solution, preferably pig trypsin.

The immersion of the tissue and/or organ in the enzymatic solution takes place for at least 12 hours, preferably from 12 to 24 hours.

The fabric treated with the pre-treatment a1)-a2) ? preferably Skin (dermo-epidermal tissue), De-Epidermised Derma (Ded).

Preferably the pre-treatment a1)-a2) ? the decellularization method described in WO2009050571.

Examples

All the experiments described here were performed with the following storage solution:

? Glycerol 0.25%

? Polyquaternium 0.004%

? EDTA 0.01%

? Poloxamer 0.18%

? Hydroxyethylcellulose (q.b)

Quantity: 10-30 ml tissue sample solution.

Glycerol: 25 ?l in 10 ml and 75 ?l in 30 ml.

Storage at room temperature of de-epidermalized Derma (Ded), previously subjected to a decellularization method, to induce both the total removal of the cellular component and its storage at room temperature.

The De-Epidermised Dermis (Ded) obtained from the upper or lower limbs of a donor has a greater cellular component than the Ded obtained from the donor trunk. For this reason, the decellularization method currently used for Ded obtained from upper or lower limbs, described in WO2009050571, is not? capable of completely removing the cellular component. , For this reason the Ded obtained from upper or lower limbs cannot? currently being considered as a starting tissue to be decellularized and used as a scaffold in the clinical setting.

The Ded obtained from the upper or lower limbs of a donor? was subjected to the method described in WO2009050571 with the addition of a further step of maintenance in solution for 1 month, in order to induce the removal of the residual cellular component and therefore obtain totally decellularised Ded. In order to evaluate whether the decellularized Ded is able to maintain its characteristics over time, analyzes were also carried out after 1-2 years of storage in solution.

The Ded ? was then kept in solution for:

? 1 month, in order to evaluate cell removal and, consequently, the production of totally decellularized Ded (Ded DEC 1 month in SS)

? 1-2 years, in order to evaluate over time the maintenance of the characteristics of the totally decellularized Ded.

Once the experimental times are over, the fabric? been subjected to the following analyses:

? Histological analysis and H&E staining

? Vitality analysis mobile phone

? Microbiological analysis: plate culture and LAL test

Results Experimentation

Histological analysis and H&E staining

As expected, Ded presents numerous cells mainly present in the skin appendages (Fig.1, upper left panel). The application of the decellularization method? object of the patent application WO2009050571 - on the Ded it allows a good removal of the cellular component which does not result for? be total (Fig. 1, top central panel). The partially decellularized Ded (Ded DEC) kept in the solution of the invention for 1 month loses completely its residual cellular component, keeping at the same time its structural characteristics unaltered (Fig. 1, top right panel). Partially decellularized Ded (Ded DEC) kept in solution for 1 year (Fig. 1, left panel, bottom) and 2 years (Fig. 1, right panel, bottom) did not show a residual cellular component as expected and maintains structural characteristics compatible with clinical use.

Histological analysis and staining with Masson's trichrome

The specific staining of the samples with Masson?s trichrome identifies a maintenance of the collagen fibers of the Ded DEC compatible with the clinical use up to 1-2 years of conservation in solution (Fig.2).

Histological analysis and Weigert staining

The specific staining of the samples with Weigert identifies the maintenance of the elastic fibers of the Ded DEC up to 1-2 years of storage in solution (Fig.3).

Vitality analysis mobile phone

The analysis of the vitality? cell phone, performed by means of the MTT test, identifies that the vitality? cell of Ded is reduced in partially decellularized Ded (Ded DEC) following the application of the decellularization method of WO2009050571 (Fig.4). Vitality? cell of Ded DEC is completely removed after 1 month of maintenance of tissue in solution (sample: Ded DEC 1 month in SS, solution) and, as expected, remains after 1-2 years of tissue in solution (samples: Ded DEC 1 aa in SS and Ded DEC 2 aa in SS) (Fig.4).

Microbiological analysis

Plate culture: microbiological analysis? was carried out on portions of Ded (1 cm<2>) partially decellularized (Ded DEC) and Ded partially decellularized and stored in solution for 1-2 years (Ded DEC 1 year in solution and Ded DEC 2 years in solution), incubated at 37°C on COS and Sabouraux plates, for 3 and 14 days respectively, in order to evaluate the growth of bacteria and/or fungi. Microbiological analysis identified the absence of bacterial and fungal contamination in the partially decellularized Ded (Ded DEC) and in the partially decellularized Ded stored for 1-2 years in LAL test solution: the LAL test ? was carried out to evaluate the possible presence of bacterial endotoxins, released by the tissue, in the storage liquid. The results show that it is not the presence of bacterial endotoxins was identified in the solution used to preserve the decellularized Ded for 1-2 years.

Cytotoxicity analysis on primary cultures of human fibroblasts

The analysis of cytotoxicity? mobile phone ? was carried out by treating primary cultures of human fibroblasts (hFs) with an extract obtained from incubation for 3 days at 4°C of decellularized Ded, kept in solution for 1-2 years, with the culture medium RPMI 1640 1% antibiotics, in a tissue/RPMI ratio of 25cm<2>/100 mL. The cells were treated with the extract for 2, 24 and 72h and were subsequently subjected to MTT tests to evaluate their viability. (samples: SS 1aa, SS 2aa). The results demonstrate that the extract is not toxic to cells (Fig.5).

Repopulation

The capacity? restocking of Ded DEC stored 1-2 years in solution? was carried out by placing in vitro culture on it, previously washed and dried on gauze, primary cultures of human fibroblasts. The essay MTT identifies a capacity? restocking of Ded DEC stored 1-2 years in solution with viable cells (samples: Ded DEC 1aa in hFs solution, Ded DEC 2aa in hFs solution) comparable to the control, i.e. cryofrozen Ded DEC (sample: Ded DEC CRIO hFs) ( Fig.6). ? in fact, it should be emphasized that the greater vitality? identified in the sample Ded DEC CRIO ? to be attributed to its partial decellularization and therefore to the maintenance of viable cells in the initial sample subjected to repopulation.

Mechanical tests

Were the mechanical tests carried out at ? Surgical Sciences and Technologies? ? Rizzoli Orthopedic Institute, Bologna ? using clinically validated equipment. In particular, the mechanical tests were carried out on Ded DEC stored for 2 years in solution, in order to evaluate its properties? biomechanics. Derma DEC cryopreserved ? was used as a control. In particular, the thickness, width, length and area (mm<2>) of each sample were measured using a digital caliper. Tensile tests were performed by clamping the lower and upper part of the specimens to the fixed lower handle and the movable upper handle of the tensile test apparatus. For each sample, the maximum load (peak load), the tensile strength (peak stress), the elastic modulus of Young (Young?s modulus) and the stiffness were measured. (slope).

The results demonstrate that the Ded DEC kept for 2 years in solution has a significantly lower (p<0.5) than the cryopreserved Derma DEC (Fig.7), a significantly lower tensile strength (p<0.5) than the Derma Cryopreserved DEC (Fig.8), a Young's elastic modulus comparable to the cryopreserved Derma DEC (Fig. 9) and a rigidity? significantly higher (p<0.5) than the cryopreserved Derma DEC (Fig.10).

Experimental conclusions

The solution ? capable of removing the residual cellular component from the Ded previously subjected to the decellularization method, maintaining its integrity? structural compatible with? clinical use and sterility? after 1-2 years. The decellularized Ded so? preserved it does not appear to be cytotoxic, it repopulates easily and, from a macroscopic analysis, it would seem to maintain its properties? biomechanical, although specific mechanical tests are needed to support these considerations.

These results allow us to hypothesize an expansion of the availability? of decellularized dermis in those cases in which the dermis, which in any case remains the preferred starting tissue, is not sufficiently available to satisfy the growing clinical demands.

Does the Ded DEC kept in solution for 2 years have some properties? lower biomechanics (maximum load, tensile strength), others comparable (Young's elastic modulus) and others higher (stiffness) than those of cryopreserved Derma DEC.

Claims (15)

A method for de-cellularizing a tissue and/or organ comprising: a) immersing an isolated tissue and/or organ in an aqueous solution comprising: at least one polymer containing at least one positively charged quaternary ammonium, at least one antiseptic agent selected from polyhexaalkylenebiguanidine and/or glycerol, and at least one surfactant; b) keeping the tissue and/or organ in the aqueous solution for at least 1 hour. 2. Method according to claim 1, wherein the tissue and/or organ ? kept in the aqueous solution for at least 1 hour, preferably for at least 2 hours, preferably for at least 3 hours, preferably for a time between 1 hour and 30 days, preferably between 2 hours and 20 days, preferably between 10 and 20 days or between 10 and 25 days. 3. A method according to claim 1 or 2, wherein the fabric is chosen from: Skin (dermo-epidermal tissue), De-epidermalized Derma (Ded). 4. Method according to claim 3, wherein the fabric is underwent a decellularization treatment. 5. A method according to any one of claims 1 to 4, wherein said polymer containing at least one positively charged quaternary ammonium? a polyquaternium, preferably ? chosen from any of the polyquaternium polyquaternium-1 to polyquaternium-48, plus? preferably any of polyquaternium 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof. 6. A method according to any one of claims 1 to 5, wherein said polyhexaalkylenebiguanidine is choice between: polyhexaethylenebiguanidine and polyhexaethylenebiguanidine. 7. A method according to any one of claims 1 to 6, wherein said glycerol is included in the solution in quantity? from 0.1% to 1% by weight, preferably from 0.4% to 0.8% by weight. 8. A method according to any one of claims 1 to 7, wherein said surfactant is a nonionic, hydrophilic surfactant, preferably a poloxamer or a mixture of poloxamers. The method according to any one of claims 1 to 8, wherein the aqueous solution further comprises a stabilizing and emulsifying agent, preferably hydroxyalkylcellulose, and/or a chelating agent, preferably the sodium salt of EDTA. 10. A method according to any one of claims 1 to 9, wherein before step a) the tissue and/or organ ? subjected to: a1) immersion in an enzymatic solution capable of engulfing at least in part fibroblasts, macrophages, mast cells, and other cells responsible for immune reactions and rejection in homologous and heterologous transplants; a2) irradiation of the tissue cos? treated with ionizing electromagnetic radiation, preferably having a frequency between 10<19 > and 10<22 >Hz. 11. A method according to claim 10, wherein the enzyme solution is a trypsin solution, preferably pork trypsin. 12. Method according to claim 10 or 11, wherein the immersion in the enzymatic solution takes place for at least 12 hours, preferably from 12 to 24 hours. 13. An aqueous composition comprising at least one polymer containing at least one positively charged quaternary ammonium, at least one antiseptic agent selected from polyhexaalkylenebiguanidine and/or glycerol and at least one surfactant, wherein said polymer is a polyquaternium, preferably selected from any of the polyquaternium polyquaternium-1 to polyquaternium-48, plus? preferably any of polyquaternium 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof; and said at least one surfactant? a nonionic, hydrophilic surfactant, preferably a poloxamer or a mixture of poloxamers. 14. Aqueous composition according to claim 13, further comprising a stabilizing and emulsifying agent, preferably hydroxyalkylcellulose, and/or a chelating agent, preferably the sodium salt of EDTA. 15. Use of the aqueous composition according to claim 13 or 14 for decellularising an isolated tissue and/or organ, possibly already? previously subjected to decellularization.