WO2023275819A1

WO2023275819A1 - Room temperature method of preservation of tissues for clinical use

Info

Publication number: WO2023275819A1
Application number: PCT/IB2022/056114
Authority: WO
Inventors: Elena Bondioli; Valeria PURPURA; Davide Melandri
Original assignee: Azienda Unita' Sanitaria Locale Della Romagna
Priority date: 2021-07-01
Filing date: 2022-06-30
Publication date: 2023-01-05
Also published as: IT202100017396A1

Abstract

The present invention relates to a method of preserving a tissue, preferably at room temperature, wherein the tissue is preserved for a period of up to 3 years in a solution comprising: at least one polymer containing at least one positively charged quaternary ammonium, at least one antiseptic agent chosen from polyhexaalkylene biguanidine and/or glycerol, and at least one surfactant. The tissue preserved with the method of the invention is chosen from: skin tissue or skin, De-epidermized Dermis (Ded), Decellularized Dermis (Derma DEC or Ded DEC), a placental tissue or a placental tissue derivative, preferably amniotic membrane, preferably human (HAM), a musculoskeletal tissue, preferably chosen from: tendons, bones, cartilage, nerves, an eye tissue, preferably chosen from: cornea and eyeball, a tissue chosen from: a valve, a vessel, aorta and an artery, preferably a femoral artery, a tissue belonging to the male and/or female genital apparatus, preferably chosen from foreskin, ovary, uterus.

Description

ROOM TEMPERATURE METHOD OF PRESERVATION OF TISSUES FOR CLINICAL USE

Technical field

The present invention relates to a method for preserving human and animal tissues intended for clinical use at room temperature.

Prior art

Tissues for use in transplantology are presently preserved with the method of 5 cryofreezing in nitrogen vapours. Notwithstanding its validity, the cryofreezing method entails high costs for the management of the cryobank, in which the tissue is stored, and requires both thawing of the tissue at the tissue bank prior to clinical use, and the use thereof at the requesting facility within and no later than 72 hours after thawing. This time limit for the use of the cryopreserved tissue, once thawed, 10 represents a major practical limit for the clinician.

Preserving tissues at room temperature is certainly preferable given the limited costs and ease of use in a clinical setting.

One method for preserving tissues at room temperature provides for the lyophilization thereof. However, this method has the disadvantage of needing to 15 rehydrate the tissue prior to use, which, in any case, does not restore the total viability and structure.

There are known techniques for the preservation, at room temperature, of tissues intended for transplantation which provide for the use of an alcohol- or glycerol- based preservation solution. Known systems of this type are for example:

20 · Dermacell®, which is an acellular dermal matrix of human origin processed using LifeNet Health (LNH) MATRACELL® technology and is preserved in Preservon®, a glycerol-based solution patented by LNH (modified Dulbecco’s solution) for the preservation of fresh decellularized, sterile, totally hydrated tissues at room temperature;

25 · AlloPatch Pliable®: it is an acellular dermal matrix of human origin immersed in an alcohol-based solution (70% ethanol).

These preservation solutions contain glycerol in a high concentration (e.g. >50%) and/or preservatives that must be eliminated by thorough washing before the tissue can be used for transplantation, since glycerol in high concentrations, like preservatives, can be toxic. Dermacell® can be used to preserve dermis intended for wound healing, but is not authorised for the preservation of dermis for use in healing burns. Furthermore, the two systems indicated are commercial tissues of American origin sold in their respective preservation solutions, which can be sold in Europe only if managed by a Tissue Bank that certifies the product’s suitability. The present invention aims to overcome the well-known drawbacks in the sector by providing a solution for preserving tissues intended for clinical use which combines the convenience of room temperature with maintenance of the necessary bioactivity of the tissue and the use of substances that do not require thorough washing prior to transplantation, as they are not toxic.

Summary of the invention

In a first aspect, the present invention relates to a method for preserving, preferably at room temperature, an isolated tissue intended for clinical use, that is, for transplantation in a patient who needs it, wherein the method comprises a step of immersing a tissue 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.

In a second aspect, the invention relates to the aqueous solution comprising the ingredient listed above and the use thereof for preserving tissues and/or organs.

In a third aspect, the invention relates to the isolated tissue preserved in the aqueous solution described above and a container containing the tissue immersed in the preservation solution according to the invention.

Brief description of the drawings

Fig. 1 shows the results of the histological analysis and H&E staining of the Derma DEC at time 0 (A), after 1 year (B), after 2 years (C), after 3 years (D) and after 4 years (E) of preservation in the solution of the invention.

Fig. 2 shows the results of the histological analysis and Masson’s trichrome staining of the Derma DEC at time 0 (A), after 1 year (B), after 2 years (C), after 3 years (D) and after 4 years (E) of preservation in the solution of the invention. Fig. 3 shows the results of the histological analysis and Weigert staining of the Derma DEC at time 0 (A), after 1 year (B), after 2 years (C), after 3 years (D) and after 4 years (E) of preservation in the solution of the invention.

Fig. 4 shows the results of the analysis of cell viability of the Derma DEC maintained in the solution of the invention for 1 -2 years.

Fig. 5 shows the results of the cytotoxicity analysis on a primary culture of human fibroblasts of an extract obtained from the incubation, for 3 days at +4°C, of the Derma DEC maintained in the solution for 1-2 years.

Fig. 6 shows the results of the cytotoxicity analysis on a primary culture of human keratinocytes of an extract obtained from the incubation, for 3 days at +4°C, of the Derma DEC maintained in the solution for 1-2 years.

Fig. 7A shows data regarding the release of the factor FGFb by the Derma DEC after 1 and 2 years of preservation in the solution.

Fig. 7B shows data regarding the release of the factor VEGF by the Derma DEC after 1 and 2 years of preservation in the solution.

Fig. 8 shows the repopulating capacity of the Derma DEC preserved for 1-2 years in the solution of the invention.

Fig. 9 shows the peak load capacity of the Derma DEC maintained for 1-2 years in the solution of the invention compared to a cryopreserved Derma DEC; Fig. 10 shows the tensile strength (peak stress) of a Derma DEC maintained for 1- 2 years in the solution of the invention compared to a cryopreserved Derma DEC; Fig. 11 shows Young’s elastic modulus of a Derma DEC maintained for 1-2 years in the solution of the invention compared to a cryopreserved Derma DEC;

Fig. 12 shows the rigidity (slope) of a Derma DEC maintained for 1-2 years in the solution of the invention compared to a cryopreserved Derma DEC;

Fig. 13 shows the results of the histological analysis and FI&E staining of an amniotic membrane at time 0 (A), after 1 year of preservation in the solution of the invention with 10X (B) and 20X (C) magnification.

Fig. 14 shows the results of the histological analysis and Masson’s trichrome staining of the amniotic membrane at time 0 (A) and after 1 year of preservation in the solution of the invention (B).

Fig. 15 shows the results of the histological analysis and Weigert staining of the amniotic membrane at time 0 (A) and after 1 year of preservation in the solution of the invention (B). Fig. 16 shows the results of the analysis of cell viability of the amniotic membrane after 1 year of preservation in the solution of the invention.

Fig. 17 shows the results of the cytotoxicity analysis on a primary culture of human fibroblasts (hFs) and FlaCaT immortalized human keratinocytes of an extract obtained from the incubation, for 3 days at +4°C, of the amniotic membrane maintained in the solution for 1 year.

Figs. 18A and B show the bioactivity, in terms of release of the growth factors bFGF and VEGF, of the amniotic membrane after preservation for 1 year in the solution of the invention.

Fig. 19 shows the results of the histological analysis and FI&E staining of the Ded DEC at time 0 (A), after 1 month (B), after 1 year (C), after 2 years (D) and after 3 years (E) of preservation in the solution of the invention.

Fig. 20 shows the results of the histological analysis and Masson’s trichrome staining of the Ded DEC at time 0 (A), after 1 month (B), after 1 year (C), after 2 years (D) and after 3 years (E) of preservation in the solution of the invention.

Fig. 21 shows the results of the histological analysis and Weigert staining of the Ded DEC at time 0 (A), after 1 month (B), after 1 year (C), after 2 years (D) and after 3 years (E) of preservation in the solution of the invention.

Fig. 22 shows the results of the analysis of cell viability of the Ded DEC after 1 month and after 1 year of preservation in the solution of the invention.

Fig. 23 shows the results of the cytotoxicity analysis on a primary culture of human fibroblasts (hFs) and FlaCaT immortalized human keratinocytes of an extract obtained from the incubation, for 3 days at +4°C, of the Ded DEC maintained in the solution for 1 year.

Fig. 24 shows the repopulating capacity of the Ded DEC preserved for 1 year in the solution of the invention;

Fig. 25 shows the peak load capacity of a Ded DEC maintained for 1 year in the solution of the invention compared to the cryopreserved Derma DEC;

Fig. 26 shows the tensile strength (peak stress) of a Ded DEC maintained for 1 year in the solution of the invention compared to the cryopreserved Derma DEC; Fig. 27 shows Young’s modulus of a Ded DEC maintained for 1 year in the solution of the invention compared to the cryopreserved Derma DEC;

Fig. 28 shows the rigidity (slope) of a Ded DEC maintained for 1 year in the solution of the invention compared to the cryopreserved Derma DEC. Detailed description of preferred embodiments of the invention

The method of preserving a tissue according to the invention comprises: a) immersing an isolated tissue 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) maintaining the tissue in the aqueous solution for a desired period, preferably up to 3 years.

The preservation method is preferably a method of preserving at room temperature. In the context of the present invention, room temperature means a temperature exceeding the freezing point of the aqueous solution, preferably between 5^eC and 35^eC, more preferably between 10^eC and 30^eC, even more preferably between 15^eC and 25^eC or between 18^eC and 25^eC. The ideal temperature for maintaining the container comprising the aqueous solution and the tissue is between 18^eC and 23^eC, preferably between 20^eC and 23^eC.

The tissue that can be preserved with the method of the invention is chosen from:

• Skin tissue or skin: consisting of the epidermal layer containing all of the cellular components making it up and a thin layer of the underlying dermis;

• De-epidermized Dermis (Ded): consisting of dermis without the overlying epidermal layer;

• Decellularized Dermis (Derma DEC or Ded DEC): consisting of dermis without the overlying epidermal layer, subjected to a decellularization method, such as the one described, for example, in W02009050571 ;

• a placental tissue or a placental tissue derivative (for example amniotic membrane, preferably human (HAM));

• a musculoskeletal tissue, for example, tendons, bones, cartilage, nerves;

• an eye tissue, for example, cornea and eyeball;

• a tissue chosen from: a valve, a vessel, aorta and an artery, preferably a femoral artery;

• a tissue belonging to the male and/or female genital apparatus (for example, foreskin, ovary, uterus). The tissues preserved with the method of the invention are isolated from a human or animal body. Typically, the tissue is removed, with the informed consent of the donor (if of human origin) in order to be subsequently implanted, following preservation with the method of the invention, in the same subject (autologous implant) or in a subject other than the donor (homologous or heterologous transplant).

The tissue that may be preserved with the method of the invention is both of human origin and of animal origin. The tissue that may be preserved with the method of the invention is tissue “as such” or decellularized tissue, i.e. tissue subjected to decellularization methods, for example the method described in W02009050571 .

In one embodiment, the tissue immersed in the solution is kept inside a container, preferably a container suitable for the preservation of biological material, for example a flexible plastic bag.

The tissue is fully or partially immersed in the preservation solution. Therefore, the amount of solution to be used will depend on the dimensions of the tissue to be preserved. The container containing the tissue fully or partially immersed in the aqueous solution is preferably hermetically closed, i.e. the container is sealed.

The hermetically sealed container is preferably inserted into a second container suitable for accommodating the container containing the tissue fully or partially immersed in the aqueous solution. The second container is preferably an overpack of a larger size than the first container.

The second container is placed under vacuum and closed, preferably by hermetic sealing.

In the preservation solution, the polymer containing at least one positively charged quaternary ammonium is a polyquaternium. Polyquaternium is the INCI (International Nomenclature for Cosmetic Ingredients) name used to describe numerous polycationic polymers belonging to different chemical classes and having in common the presence of at least one quaternary ammonium, preferably several quaternary ammonium groups. Within the definition of “polyquaternium” the INCI includes at least 40 different polymers, which are indicated with a sequential numbering assigned based on the order of registration and not based on their chemical nature.

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

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

Preferably, the polyquaternium used in the present preservation solution is chosen from any one of polyquaterniums from polyquaternium-1 to polyquaternium-48, i.e. from any one of polyquaterniums from 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof. In one embodiment, the polyquaternium used in the present solution is a mixture of two or more polyquaterniums chosen from polyquaterniums 1 to 48.

The correspondence between the numbering of the polyquaterniums and the respective chemical names is known and is thus not entirely described herein.

The polymer containing at least one positively charged quaternary ammonium is present in the solution in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight.

The at least one antiseptic agent is chosen from: polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine or polyhexayethylene biguanidine, glycerol and a combination thereof.

The at least one antiseptic agent is present in the solution in an amount of 0.0001% to 1%, preferably 0.0001% to 0.8% by weight.

If the antiseptic agent is glycerol, it is preferably included in the solution in an amount of 0.1% to 1% by weight, preferably 0.4% to 0.8% by weight.

Alternatively, the at least one antiseptic agent is present in the solution in an amount of 0.0001% to 0.005% by weight, preferably 0.00015% to 0.003% by weight. The at least one surfactant is preferably a non-ionic, hydrophilic surfactant. The surfactant is preferably a poloxamer. Poloxamers are a class of non-ionic polymeric surfactants with a triblock structure comprising a central hydrophobic chain of propylene glycol bound at the sides to two hydrophilic chains of polyethylene glycol (PEG). An example of a poloxamer that can be used in the solution of the invention is poloxamer 407, which has a PEG chain with a length of about 100 repeat units and a propylene glycol block with a length of about 56 repeat units. The at least one surfactant is present in the aqueous solution in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight.

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

The chelating agent is preferably chosen from: EDTA and/or salts thereof, preferably EDTA sodium salt, and polyamine acetylacetonate. The amount of chelating agent present in the solution is 0.005% to 0.5% by weight, preferably 0.01% to 0.1% by weight.

The at least one stabilizing and emulsifying agent, if present, is chosen from hydroxyalkyl cellulose, preferably hydroxymethyl cellulose.

The at least one stabilizing and emulsifying agent is present in the solution in the amount necessary (q.s., quantum sufficit) to obtain an adequate emulsion of the ingredients.

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

In one embodiment, the aqueous solution comprises: polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine in an amount of 0.0001% to 0.005% by weight, preferably 0.00015% to 0.003% by weight, at least one polyquaternium from 1 a 48 or a mixture of polyquaterniums in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight and at least one surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight. In one embodiment, the aqueous solution comprises glycerol in an amount of 0.1% to 1% by weight, preferably 0.4% to 0.8% by weight, at least one polyquaternium from 1 to 48 or a mixture of polyquaterniums in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight, and at least one surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight.

In one embodiment, the aqueous solution comprises: polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine, or glycerol, at least one polyquaternium from 1 to 48 or a mixture of polyquaterniums, at least one surfactant, preferably a poloxamer or a mixture of poloxamers, hydroxyalkyl cellulose, preferably hydroxymethyl cellulose, and/or EDTA.

In a preferred embodiment, the aqueous preservation solution comprises:

• polyhexaalkylene biguanidine, preferably, polyhexamethylene biguanidine, in an amount of 0.0001% to 0.005% by weight, preferably 0.00015% to 0.003% by weight, and/or glycerol in an amount of 0.1% to 1% by weight, preferably 0.4% to 0.8% by weight;

• at least one polyquaternium from 1 to 48 or a mixture of polyquaterniums, i.e. any one of polyquaterniums from 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof, in an amount of 0.001% to 0.010% by weight, preferably 0.003% to 0.006% by weight;

• a non-ionic, hydrophilic surfactant, preferably a poloxamer or a mixture of poloxamers, in an amount of 0.05% to 0.5% by weight, preferably 0.1% to 0.3% by weight;

• preferably, hydroxyalkyl cellulose, more preferably hydroxymethyl cellulose, in a sufficient amount (q.s.) to obtain an adequate emulsion of the ingredients; and/or • preferably, EDTA and/or a salt thereof, more preferably EDTA sodium salt, in an amount of 0.005% to 0.5% by weight, preferably 0.01% to 0.1% by weight.

The presence of glycerol in the solution has numerous advantages. In particular, glycerol possesses numerous properties and is also present in nature (vegetable glycerol): it is hydrating and acts as a powerful antibacterial and antiviral agent; therefore, it assures the sterility of the final product stored at room temperature. Furthermore, it is more effective from a biochemical viewpoint compared to the classic antiseptics/antimicrobials used in cosmetic formulations.

Glycerol is already used to preserve tissues, in particular soft and musculoskeletal tissues; however, it is used in high concentrations, for example > 50%. In these amounts glycerol can be toxic for cells, thus negatively affecting the physiological properties of the tissue itself. For this reason, tissue preserved in glycerol, for example the glycerol-preserved skin distributed by some Italian skin banks, must undergo numerous washes prior to its clinical use.

The Applicant has found that by using glycerol in reduced amounts (maximum 1% by weight) in a mixture with at least one polymer containing at least one positively charged quaternary ammonium and at least one surfactant, one may achieve tissue preservation without the manifestation of negative effects due to the possible toxicity of high concentrations of glycerol and the need wash the tissue preserved in glycerol repeatedly prior to the use thereof. The same effect is also obtained with an aqueous solution in which polyhexaalkylene biguanidine, preferably polyhexamethylene biguanidine, is used in place of glycerol.

The preservation method of the invention enables tissue to be preserved for a period equal to or less than 3 years, preferably equal to or less than 2 years, more preferably for a period of between 7 days and 2 years, even more preferably for a period of between 30 days and 2 years. The Applicant has verified, through the experiments included in the present patent application, that a tissue preserved in the solution of the invention for up to 2 years maintains its morphological and structural characteristics intact and good bioactivity. Bioactivity means the ability of tissue to release growth factors such as FGFb and VEGF. The repopulating capacity of the tissue, understood as the ability of viable cells to regrow, remains optimal up to 2 years of storage.

The mechanical properties of the tissue are also preserved intact.

Furthermore, the tissue remains sterile up to 2 years, i.e. free of bacterial or fungal contamination and free of bacterial endotoxins and is not cytotoxic.

The purpose of tissue preservation is to perform a transplantation of that tissue in an individual who needs it. That individual can be the same subject from whom the tissue was taken (autologous implant) or a different subject (homologous or heterologous transplant).

The invention also relates to a tissue partially or fully immersed in an aqueous preservation solution as described above, as well as a container comprising the tissue as described above, partially or fully immersed in the aqueous solution as described above.

Therefore, the invention relates to a composition comprising a tissue and an aqueous preservation solution as described above, wherein the tissue is partially or fully covered by the solution.

The container comprising the tissue immersed in the aqueous preservation solution is preferably a container suitable for preserving biological material, for example a flexible plastic bag. Said container is preferably closed with a hermetic seal.

The container is inserted into a second container suitable for accommodating the container containing the tissue immersed in the aqueous solution. The second container is a preferably an overpack of larger size than the first container.

The aqueous solution is present in the container containing the tissue so as to partially, preferably completely, cover the tissue. Therefore, the amount of solution present in the container will depend on the dimensions of the tissue to be preserved.

The invention also relates to an aqueous preservation solution as described above and the use thereof to preserve a tissue as described above. Examples

All of the experiments described herein were carried out with the following preservation solution: · Glycerol 0.25%

• Polyquaternium 0.004%

• EDTA O.01%

• Poloxamer 0.18%

• Hydroxyethyl cellulose (q.s.) Amount: 10-30 ml solution per tissue sample.

Glycerol: 25 mI in 10 ml and 75 mI in 30 ml.

Experiment 1: “Preservation of Decellularized Dermis (Derma DEC) at room temperature” The Derma DEC was introduced into bags for packaging, to which the preservation solution according to the invention was subsequently added.

The bag containing the Derma DEC and the preservation solution was then introduced inside overpacks placed under vacuum and sealed. After 1 -2 years of storage of the Derma DEC in the solution, the following analyses were performed:

• Morphological analysis

• Histological analysis and H&E, Masson’s trichrome and Weigert staining

• Analysis of cell viability

• Microbiological analysis: plate culture and LAL test · Cytotoxicity analysis on primary cultures of fibroblasts and cell lines

• Bioactivity

• Repopulation

• Mechanical tests

Results of Experiment 1: “Preservation of Derma DEC at room temperature”

The analyses and results obtained with regard to experiment 1 are reported below:

Morphological analysis The Derma DEC maintained in the solution for 1 and 2 years retains morphological characteristics similar to those of thawed tissue.

Histological analysis and H&E staining

The Derma DEC maintained in the solution for 1 , 2 and 3 years retains structural characteristics compatible with clinical use (Fig. 1 B-D)

The Derma DEC maintained in the solution for 4 years appears to have deteriorated; the structural characteristics maintained are nonetheless potentially compatible with clinical use (Fig. 1 E).

Histological analysis and Masson’s trichrome staining The specific Masson’s trichrome staining of the samples identifies a maintenance of the collagen fibres of the Derma DEC - preserved in the solution for 1 , 2 and 3 years - compatible with clinical use (Fig. 2B-D).

The specific Masson’s trichrome staining of the samples identifies collagen fibres of the Derma DEC - preserved in the solution for 4 years - which have deteriorated. The structural characteristics maintained are nonetheless potentially compatible with clinical use (Fig. 2E).

Histological analysis and Weigert staining

The specific Weigert staining of the samples identifies a loss of the elastic fibres of the Derma DEC preserved in the solution for 1 -4 years (Fig.3).

Analysis of cell viability

The analysis of cell viability, performed by means of an MTT assay, was carried out in order to demonstrate the total removal of the cellular component. It should be considered that the decellularization method applied to the Derma is already capable of removing cells. The analysis applied on Derma DEC maintained in the solution for 1-2 years is thus only an analysis to confirm cell removal also in the tissues under examination. As expected, the Derma DEC maintained in the solution for 1-2 years shows the removal of the cellular component Fig. 4). Microbiological analysis

Plate culture : the microbiological analysis was performed on portions of Derma DEC (1cm²) preserved in the solution for 1-2 years, which were incubated at 37°C on COS and Sabouraud plates for 3 and 14 days, respectively, to determine whether there was any growth of bacteria and/or fungi. The microbiological analysis identified the absence of bacterial and fungal contamination after 1-2 years of preservation of the Derma DEC in the solution. LAL test : the LAL test was carried out to evaluate the possible presence of bacterial endotoxins, released by the tissue, in the preservation liquid. The results show that no bacterial endotoxins are present in the solution used to preserve the Derma DEC for 1 -2 years.

Cytotoxicity analysis on a primary culture of human fibroblasts and on a cell line of human keratinocytes

The cell cytotoxicity analysis was performed by treating:

• a primary culture of human fibroblasts (hFs)

• the immortalized human keratinocyte cell line HaCaT with an extract obtained from 3 days of incubation, at +4°C, of the Derma DEC, maintained in the solution for 1-2 years, with RPMI 1640 culture medium + 1% antibiotics, in a Derma DEC/RPMI ratio of 25cm²/100 ml. The cells were subsequently treated with the extract for 2, 24 and 72h and were then subjected to an MTT assay to evaluate their viability (samples: SS 1 yr and SS 2yr). Use was made of an extract obtained from cryofrozen Derma DEC which was thawed and immediately immersed in RMP1 1640, as a control (CRYO sample). Untreated cells maintained in RPMI 1640 culture medium + 1% antibiotics in the presence or absence of foetal bovine serum, were used as a control of cell viability and growth over the times of the analysis (SERUM and NO SERUM samples). The results demonstrate that the extract is not toxic for cells (Fig.5-6).

Bioactivity

An analysis of the bioactivity of the Derma DEC preserved for 1-2 years in the solution was conducted by incubating the tissue for 3 days at +4°C with RPM1 1640 culture medium + 1% antibiotics, in a Derma DEC/RPMI ratio of 25cm²/100 ml. The medium incubated with the tissue was then centrifuged after 3 days and subjected to an ELISA assay in order to identify the release of the growth factors FGFb and VEGF, which are important for tissue regeneration and angiogenesis.

The results demonstrate that:

• The Derma DEC releases the growth factor FGFb at comparable levels after 1 and 2 years of preservation in the solution. The amount of FGFb released by the Derma DEC after 1-2 years of preservation in the solution is about 40% lower compared to its release by the cryofrozen Derma DEC. The lyophilized Derma DEC shows a release of FGFb that is considerably lower compared to the other three samples (Fig 7 A). • The Derma DEC preserved in the solution releases the growth factor VEGF at comparable levels after 1 and 2 years. The amount of VEGF released by the Derma DEC after 1-2 years of preservation in the solution is comparable to its release by the cryopreserved and lyophilized Derma DEC (Fig 7 B).

The Derma DEC preserved in the solution thus maintains bioactive capacities.

Repopulation

The repopulating capacity of the Derma DEC preserved for 1 -2 years in the solution was analysed by culturing primary cultures of human fibroblasts on it in vitro, after it had been previously washed and dried on gauze. The MTT assay identifies a repopulating capacity of the Derma DEC in vitro with viable cells that is comparable to that of the control, i.e. cryofrozen Derma DEC (Fig. 8).

Mechanical tests

The mechanical tests were performed at the “Surgical Sciences and Technologies” department of the Rizzoli Orthopaedic Institute in Bologna using instruments validated for clinical use. In particular, the mechanical tests were carried out on Derma DEC preserved for 1-2 years in the solution, in order to assess its biomechanical properties. The cryopreserved Derma DEC was used as a control. In particular, the thickness, width, length and area (mm²) of every sample were measured using a digital calliper. The tensile tests were carried out by securing the lower and upper part of the samples to the lower fixed grip and upper movable grip of the tensile testing apparatus. The peak load, tensile strength (peak stress), Young’s elastic modulus and rigidity (slope) were measured for each sample.

The results demonstrate that the Derma DEC maintained for 1-2 years in the solution has a peak load capacity that is comparable to (at 1 year) or significantly higher (p<0.5) (at 2 years) than that of the cryopreserved Derma DEC (Fig. 9), a tensile strength that is comparable to that of the cryopreserved Derma DEC (Fig. 10), a Young’s elastic modulus that is significantly higher (p<0.5) than that of the cryopreserved Derma DEC (Fig. 11 ) and a rigidity that is comparable to (at 1 year) or significantly higher (p<0.5) (at 2 years) than that of cryopreserved Derma DEC (Fig. 12). It should be considered that the enzymatic-physical method used for decellularization is capable of enhancing, over time, some biological parameters, including peak load and rigidity. Experiment 1 conclusions

The preservation solution of the invention is capable of maintaining the structural integrity and sterility of the Derma DEC up to 2 years of preservation. The Derma DEC thus preserved is not cytotoxic, easily repopulates and, based on a macroscopic analysis, seems to maintain its biomechanical properties, though specific mechanical tests are necessary to support these considerations. Furthermore, the Derma DEC preserved for 1-2 years in the solution maintains, albeit partially, its bioactivity, which is in any case appreciable. The Derma DEC maintained in the solution for 1-2 years maintains or even has enhanced biomechanical properties compared to the cryopreserved Derma DEC.

Experiment 2

After processing, human amniotic membrane (HAM) was preserved in packaging bags to which the preservation solution was subsequently added. The bag containing HAM and the preservation solution was then sealed and inserted into vacuum sealed overpacks. Once the experimental period was over, the HAM was subjected to the following analyses:

• Histological analysis and H&E staining · Histological analysis and Masson ’s trichrome staining

• Histological analysis and Weigert staining

• Analysis of cell viability

• Microbiological analysis: plate culture and LAL test

• Cytotoxicity analysis on primary cultures of fibroblasts and cell lines · Bioactivity

Experiment 2 results Histological analysis and H&E staining

The histological analysis and haematoxylin and eosin staining carried out on the amniotic membrane after 1 year of preservation in the solution show that its structure was maintained intact (Fig. 13). The epithelial layer shows that the cellular component was maintained. However, some cells appear to be close to detaching from the basal layer (Fig. 13).

Histological analysis and Masson’s trichrome staining

The histological analysis and specific Masson’s trichrome staining of the samples show the maintenance of the collagen fibres of HAM preserved for 1 year in the solution (Fig. 14).

Histological analysis and Weigert staining

The histological analysis and specific Weigert staining of the samples show a comparable condition between freshly collected HAM and HAM preserved for 1 year in the solution (Fig. 15).

Analysis of cell viability

The analysis of the cell viability, performed by means of an MTT assay, was carried out to assess the maintenance of viable cells in the HAM after 1 year of preservation in the solution (sample: amniotic membrane 1 year in SS). An intermediate evaluation was also performed after 6 months of preservation in the solution (sample: amniotic membrane 6 months in SS). The HAM maintained in the solution for 6 months retains 50% of cell viability compared to freshly collected HAM (Fig.16). The HAM maintained in the solution for 1 year retains 30% of cell viability compared to the initial unprocessed HAM (Fig.12).

Microbiological analysis

Plate culture : a microbiological analysis was performed on HAM portions (1cm²), preserved in the solution for 6 months and 1 year, which were incubated at 37°C on COS and Sabouraud plates for 3 and 14 days, respectively, in order to assess the growth of bacteria and/or fungi. The microbiological analysis identified the absence of bacterial and fungal contamination after 6 months and 1 year of preservation of the HAM in the solution.

LAL test the LAL test was performed to determine the possible presence of bacterial endotoxins, released by the tissue, in the preservation liquid. The results show that no presence of bacterial endotoxins was identified in the solution used to preserve the HAM for 1 year.

Cytotoxicity analysis on primary cultures of fibroblasts and cell lines

The analysis of cell cytotoxicity was performed by treating:

• primary cultures of human fibroblasts (hFs)

• the immortalized human keratinocyte cell line HaCaT with an extract obtained from incubation, for 3 days at +4°C, of HAM maintained in the solution for 1 year, with the RPMI 1640 culture medium + 1% antibiotics, in a HAM/RPMI ratio of 25cm²/100 ml. The cells were treated with the extract for 2, 24 and 72h and were subsequently subjected to an MTT assay in order to assess their viability (samples: SS 1 yr). Use was made of untreated cells, cultured in the presence or absence of serum, as a control (samples: SERUM and NO SERUM). The results demonstrate that the extract is not toxic for the cells (Fig. 17). Bioactivity

Bioactivity was assessed by determining the release of the growth factors bFGF and VEGF, which are important for tissue regeneration and angiogenesis, from the HAM preserved for 1 year in the solution (sample: HAM SS 1 yr). The bioactivity of freshly collected HAM (sample: HAM) and cryopreserved HAM (sample: HAM CRYO) was used as a control.

The results demonstrate that:

• the release of bFGF by the HAM preserved for 1 year in the solution is comparable to the release by the cryopreserved control HAM (Fig. 18A).

• the release of VEGF by the HAM preserved for 1 year in the solution is comparable to release by the cryopreserved control HAM (Fig. 18B).

The HAM preserved in the solution of the invention thus maintains its bioactive capacities.

Experiment 2 conclusions

The preservation solution is capable of maintaining the structure and a good percentage of viable cells in HAM. However, the cells of the epithelial layer are viable but close to detaching from the basal membrane. The HAM preserved in the solution for 1 year is also sterile, is not cytotoxic and maintains its bioactive capacities.

Experiment 3 “Preservation at room temperature of De-epidermized Dermis (Ded), previously subjected to a decellularization method to induce both the total removal of the cellular component and its preservation at room temperature”

De-epidermized Dermis (Ded) has a larger cellular component than the Derma. For this reason, the decellularization method presently used for the Derma (for example the technique described in PCT/IB2008/002753) is not capable of completely removing the cellular component when applied to the Ded, which, consequently, cannot be taken into consideration as the starting tissue to be decellularized and used as a scaffold in a clinical setting.

Considering the growing clinical demand for decellularized Dermis, the Ded was subjected to the decellularization method presently used for the Derma (for example the one described in PCT/IB2008/002753), in order to obtain the Derma DEC, though as a further added step it was maintained in the preservation solution of the invention for 1 month in order to induce the removal of the residual cellular component and thereby obtain decellularized Ded. It should in any case be underscored that the Derma used for the production of Derma DEC is to be preferred as the starting tissue, as it enables a more compact decellularized scaffold to be obtained. In fact, the Ded has more skin adnexa, which, when the tissue is subjected to decellularization, are transformed into non-compact acellular zones that, as a consequence, could negatively affect the biomechanical properties of the tissue. It should be considered, however, that such regions could be more easily repopulated once grafted onto the patient. For the purpose of assessing whether the decellularized Ded is capable of maintaining its characteristics over time, analyses were also performed after 1 year of preservation in the solution of the invention.

The Ded was thus maintained in the solution for:

• 1 month, in order to assess cell removal and, consequently, the production of decellularized Ded (Ded DEC)

• 1 year, a in order to assess whether the characteristics of the decellularized Ded are maintained over time

Once the experimental period was over, the tissue was subjected to the following analyses:

_• Histological analysis and H&E staining

_• Histological analysis and Masson ’s trichrome staining

_• Histological analysis and Weigert staining

_• Analysis of cell viability

_• Microbiological analysis: plate culture and LAL test

_• Cytotoxicity analysis on primary cultures of fibroblasts and a cell line of keratinocytes • Cellular repopulation

• Mechanical tests

Experiment 3 results

Histological analysis and H&E staining

The partially decellularized Ded maintained in the solution for 1 month completely loses its residual cellular component, while at the same time maintaining its structural characteristics intact. The Ded DEC maintained in the solution for 1-2 years (Fig.19 C-D) maintains structural characteristics compatible with clinical use.

The Ded DEC maintained in the solution for 3 years is partially deteriorated; however, the structural characteristics maintained are potentially compatible with clinical use (Fig. 19 E). Histological analysis and Masson’s trichrome staining

The specific Masson’s trichrome staining of the samples identifies a maintenance of the collagen fibres of the Ded DEC compatible with clinical use after 1 month and 1-2 years of preservation in the solution (Fig. 20B-D).

The specific Masson’s trichrome staining of the samples identifies collagen fibres of the Ded DEC - preserved in the solution for 3 years - which are partially deteriorated. Flowever, the structural characteristics maintained are potentially compatible with clinical use (Fig. 20E).

Histological analysis and Weigert staining

The specific Weigert staining of the samples identifies the maintenance of the elastic fibres of the Ded DEC after 1 month and 1 year of preservation in the solution (Fig. 21 B-C).

The specific Weigert staining of the samples identifies a loss of the elastic fibres of the Ded DEC preserved in the solution for 2-3 years (Fig.21 D-E).

Analysis of cell viability The analysis of cell viability, performed by means of an MTT assay, shows that the Ded cell viability is considerably reduced in the Ded DEC following the application of the decellularization method (Fig. 22). The cell viability of the Ded DEC is wholly removed after the tissue has been maintained in the solution for 1 month (sample: Ded DEC 1 month in SS) (Fig. 22). The removal of cell viability is confirmed, as expected, after the Ded DEC has been maintained in the solution for 1 year (sample: Ded DEC 1 year in SS) (Fig. 22).

Microbiological analysis

Plate culture : the microbiological analysis was performed on portions of Ded (1 cm²) preserved in the solution for 1 month and 1 year, which were incubated at 37°C on COS and Sabouraud plates for 3 and 14 days, respectively, in order to assess the growth of bacteria and/or fungi. The microbiological analysis identified the absence of bacterial and fungal contamination after 1 month and 1 year of preservation of the Decellularized Ded in the solution.

LAL test the LAL test was performed to determine the possible presence of bacterial endotoxins, released by the tissue, in the preservation liquid. The results show that no presence of bacterial endotoxins was identified in the solution used to preserve the Decellularized Ded for 1 month and 1 year.

Cytotoxicity analysis on primary cultures of human fibroblasts and HaCaT keratinocyte cell lines

The analysis of cell cytotoxicity was performed by treating:

• primary cultures of human fibroblasts

• the immortalized human keratinocyte cell line HaCaT with an extract obtained from incubation, for 3 days at +4°C, of Decellularized Ded, maintained in the solution for 1 year, with the RPMI 1640 culture medium + 1% antibiotics, in a tissue/RPMI ratio of 25cm²/100 ml. The cells were treated with the extract for 2, 24 and 72h and were subsequently subjected to an MTT assay in order to assess their viability (sample: SS 1 yr). The results demonstrate that the extract is not toxic for the cells (Fig. 23).

Repopulation

The repopulating capacity of the Ded DEC preserved for 1 year in the solution was analysed by culturing primary cultures of human fibroblasts on it in vitro, after it had been previously washed and dried with gauze. The MTT assay identifies a good repopulating capacity of the Ded DEC in vitro with viable cells (sample: Ded DEC 1 yr in the solution + hFs) compared to the control, i.e. cryofrozen Derma DEC (sample: Derma DEC CRYO + hFs) (Fig. 24). The lyophilized derma DEC (sample: Derma DEC LYOPH + hFs) shows a lower repopulating capacity compared both to the Ded DEC maintained 1 yr in CONTACTA SOLUTION, and the cryofrozen Derma DEC (Fig. 24).

Mechanical tests

The mechanical tests were performed at the “Surgical Sciences and Technologies” department of the Rizzoli Orthopaedic Institute in Bologna using instruments validated for clinical use. In particular, the mechanical tests were carried out on Ded DEC preserved for 1 year in the solution, in order to assess its biomechanical properties. Cryopreserved Derma DEC was used as a control. In particular, the thickness, width, length and area (mm²) of every sample were measured using a digital calliper. The tensile tests were carried out by securing the lower and upper part of the samples to the lower fixed grip and upper movable grip of the tensile testing apparatus. The peak load, tensile strength (peak stress), Young’s elastic modulus and rigidity (slope) were measured for each sample.

The results demonstrate that the Ded DEC maintained for 1 year in the solution has a significantly lower (p<0.1) peak load capacity than the cryopreserved Derma DEC (Fig. 25), a significantly lower (p<0.5) tensile strength than the cryopreserved Derma DEC (Fig. 26), a significantly higher (p<0.5) Young’s elastic modulus than the cryopreserved Derma DEC (Fig. 27) and a significantly lower (p<0.5) rigidity than the cryopreserved Derma DEC (Fig. 28).

Experiment 3 conclusions

The solution of the invention is capable of removing the residual cellular component from the Ded previously subjected to the decellularization method presently used for the Derma, while maintaining the structural integrity and sterility thereof after 1 month. The solution is capable of maintaining the structural integrity and sterility of the Decellularized Ded for 1 year. The Decellularized Ded thus preserved is not cytotoxic, easily repopulates and, based on a macroscopic analysis, seems to maintain its biomechanical properties, though specific mechanical tests are necessary to support such considerations.

These results suggest the possibility of expanding the availability of Decellularized Dermis in cases where Derma, which nonetheless remains the preferred starting tissue, is not sufficiently available to satisfy the growing clinical demand. The Ded DEC maintained in the solution for 1 year has some inferior biomechanical characteristics (peak load, tensile strength, rigidity) and other superior ones (Young’s elastic modulus) compared to the cryopreserved Derma DEC.

Claims

1 . A method of preserving a tissue, preferably at room temperature, comprising: (a) Immersing an isolated tissue in an aqueous solution comprising: at least one polymer containing at least one positively charged quaternary ammonium, at least one antiseptic agent chosen from polyhexaalkylene biguanidine and/or glycerol, and at least one surfactant;

(c) Maintaining the tissue in the aqueous solution for a desired period, preferably up to 3 years.

2. Method according to claim 1 , wherein the storage period is equal to or less than 2 years, more preferably between 7 days and 2 years, even more preferably between 30 days and 2 years.

3. Method according to claim 1 or 2, wherein the tissue is chosen from: skin tissue or skin, De-epidermized Dermis (Ded), Decellularized Dermis (Derma DEC or Ded DEC), a placental tissue or placental tissue derivative, preferably amniotic membrane, preferably human (HAM), a musculoskeletal tissue, preferably chosen from: tendons, bones, cartilage, nerves, an eye tissue, preferably chosen from: cornea and eyeball, a tissue chosen from: a valve, a vessel, aorta and an artery, preferably a femoral artery, a tissue belonging to the male and/or female genital apparatus, preferably chosen from foreskin, ovary, uterus.

4. Method according to any one of claims 1 to 3, wherein said polymer containing at least one positively charged quaternary ammonium is a polyquaternium, preferably it is chosen from any one of polyquaternium-1 to polyquaternium-48, more preferably any one of polyquaternium-1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof.

5. Method according to any one of claims 1 to 4, wherein said polyhexaalkylene biguanidine is chosen from: polyhexamethylene biguanidine and polyhexaethylene biguanidine.

6. Method according to any one of claims 1 to 5, wherein said glycerol is included in the solution in an amount of 0.1% to 1% by weight, preferably 0.4% to 0.8% by weight.

7. Method according to any one of claims 1 to 6, wherein said surfactant is a non ionic, hydrophilic surfactant, preferably a poloxamer or a mixture of poloxamers.

8. Method according to any one of claims 1 to 7, wherein the aqueous solution further comprises a stabilizing and emulsifying agent, preferably hydroxyalkyl cellulose, and/or a chelating agent, preferably EDTA sodium salt or polyamine acetylacetonate.

9. An aqueous solution comprising at least one polymer containing at least one positively charged quaternary ammonium, at least one antiseptic agent chosen from polyhexaalkylene biguanidine and/or glycerol and at least one surfactant, wherein said polymer is a polyquaternium, preferably chosen from any one of polyquaterniums from polyquaternium-1 to polyquaternium-48, more preferably any one of polyquaterniums 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 48 and combinations thereof; and said at least one surfactant is a non-ionic, hydrophilic surfactant, preferably a poloxamer or a mixture of poloxamers, wherein the at least one antiseptic agent is present in the solution in an amount of 0.0001% to 1% by weight and wherein the at least one polymer containing at least one positively charged quaternary ammonium is present in the solution in an amount of 0.001% to 0.010% by weight.

10. Aqueous solution according to claim 9, wherein the at least one antiseptic agent is present in the solution in an amount of 0.0001% to 0.8% by weight and wherein the at least one positively charged quaternary ammonium is present in the solution in an amount of 0.003% to 0.006% by weight

11. Aqueous solution according to claim 9 or 10, further comprising a stabilizing and emulsifying agent, preferably hydroxyalkyl cellulose, and/or a chelating agent, preferably EDTA sodium salt or polyamine acetylacetonate.

12. Use of the aqueous solution according to any of one of claims 9 to 11 to preserve an isolated tissue, preferably for a period of up to 3 years, preferably for a period of 2 years or less, more preferably between 7 days and 2 years, even more preferably between 30 days and 2 years.

13. A container comprising an isolated tissue immersed totally or partially in the aqueous solution according to any of one of claims 9 to 11 , wherein said container is preferably hermetically sealed and is preferably a flexible plastic bag.

14. Container comprising a further container containing a tissue fully or partially immersed in the aqueous solution according to any of one of claims 9 to11 , wherein said container is preferably a plastic overpack and said further container is a plastic bag.