Method for producing decellularized tissue scaffold

Abstract

The invention relates to a method for producing a decellularized tissue scaffold. The invention also relates to a tissue scaffold produced by the method. The invention particularly relates to a pig tissue scaffold. The methods include reduced levels of anionic detergents and avoid the use of protease inhibitors of animal origin to produce tissue scaffolds with advantageous properties.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202180017668.5, with an application date of March 5, 2021 and an invention title of "A method for producing acellular tissue scaffolds". Technical Field

[0002] The present invention relates to a method for producing acellular tissue scaffolds. The present invention also relates to tissue scaffolds produced by said method. Background Art

[0003] Soft tissue grafts of animal or human origin are used in various medical procedures for surgical repair or replacement of soft tissues. For example, such grafts can be used to repair or reconstruct tissues in hernia repair, pelvic organ prolapse or ligament reconstruction.

[0004] In order for human tissue (allograft) or animal tissue (xenograft) to be used as a safe and effective tissue scaffold, it is necessary to ensure that the graft is free of bacteria and viruses, acellular, and free of DNA to prevent graft rejection, and to retain the functional properties of the natural tissue from which it is derived, including biomechanical properties and the ability to act as an extracellular matrix (ECM).

[0005] It is known in the art that, in addition to one or more protease inhibitors, the use of anionic detergents such as sodium dodecyl sulfate (SDS) is very effective in acellularizing soft tissues while maintaining their ECM structure to produce effective biologic scaffolds (EP1392372B1). However, even at relatively low concentrations, SDS is highly cytotoxic, which can lead to the failure of the biocompatibility of the graft because detergent residues remain in the graft. In addition, certain protease inhibitors, such as aprotinin, are usually of animal origin, which is associated with the risk of transplant recipients being exposed to zoonotic diseases (e.g., transmissible spongiform encephalopathies including bovine spongiform encephalopathy), which is unacceptable in the context of medical products.

[0006] Another problem with current processing methods is the effective removal of viruses from the graft itself when using non-human tissue. It is important to ensure that xenografts are biologically safe by reducing potential zoonotic pathogens (e.g., porcine parvovirus (PPV) and porcine endogenous retrovirus (PERV)) to a safe level. To this end, animal tissues must be exposed to a disinfection / sterilization process that has been shown to reduce zoonoses by up to 6-log. However, this magnitude of reduction cannot typically be achieved by traditional sterilization methods, such as gamma irradiation at a dose of 25 kGy.

[0007] The present invention aims to provide an improved method for producing acellular tissue scaffolds that overcomes or partially ameliorates the problems associated with methods known in the art. Summary of the Invention

[0008] In one aspect, the present invention relates to a method for producing a decellularized tissue scaffold, the method comprising the steps of:

[0009] · incubating the starting tissue with a defatting agent;

[0010] · incubating the tissue with an anionic detergent;

[0011] · incubating the tissue with a hypotonic solution at least once, incubating with a hypertonic solution at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0012] · incubating the tissue with a DNA removing agent to obtain a decellularized tissue scaffold.

[0013] It should be understood that the steps of the method of the present invention do not need to be carried out in the order provided above. Additionally, or alternatively, two or more steps may be combined into one step. By way of example only, the steps of incubating the tissue with a hypotonic buffer at least once and incubating the tissue with an anionic detergent may be combined into one step. Exemplary step sequences are provided elsewhere in this specification.

[0014] Furthermore, multiple steps of each incubation may be included in the method. For example, the method may include multiple steps of incubating the tissue with a defatting agent.

[0015] Suitably, the starting tissue may be selected from skin, meniscus, tendon, ligament, cartilage, muscle, blood vessel, and organ.

[0016] Suitably, the defatting agent may be a polar solvent. Suitably, the polar solvent may be acetone. Suitably, the concentration of the defatting agent may be about 80% to about 100% (v / v).

[0017] Suitably, the anionic detergent may be sodium dodecyl sulfate (SDS) or sodium deoxycholate. Suitably, the concentration of the anionic detergent (e.g., SDS or sodium deoxycholate) may be equal to or less than 0.2% (w / v).

[0018] Suitably, the method does not include protease inhibitors. Suitably, the method does not include protease inhibitors of animal origin. Suitably, the method may include protease inhibitors of non-animal origin. Suitably, the method may include metalloprotease inhibitors of non-animal origin. Suitably, the protease inhibitor of non-animal origin may be EDTA. Suitably, the concentration of the protease inhibitor of non-animal origin may be about 0.1% (w / v).

[0019] Suitably, the method further includes one or more rinsing steps, suitably after each step of cell lysis, and thus suitably, one or more rinsing steps are carried out after incubating the tissue with a hypotonic solution at least once and incubating the tissue with a hypertonic solution at least once and / or subjecting the tissue to at least one freeze / thaw cycle.

[0020] Suitably, the method does not include α-galactosidase.

[0021] Suitably, DNA removal will be achieved by using a DNA removal agent, which can be selected from the group consisting of endonucleases or other treatments that break down DNA to effect elution from the tissue. The endonuclease can be selected from human endonucleases, which are selected from DNase type I, DNase type II, DNase type III, and RNase, or other sources, including bacterially derived endonucleases or any combination of two or more thereof. Suitably, the endonuclease can be a bacterially derived endonuclease, such as a genetically engineered Serratia marcessens endonuclease, which can be selected from any commercially available endonuclease, such as Benzonase®. Suitably, the concentration of the endonuclease can be from about 0.5 to about 50 U / ml. Suitably, the concentration of the endonuclease is about 5 U / ml.

[0022] Suitably, the method can include the step of incubating the scaffold with an antimicrobial agent. Suitably, the antimicrobial agent can be an oxidizing agent. Suitably, the oxidizing agent can be peracetic acid. Suitably, the concentration of peracetic acid can be from about 500 to about 10,000 ppm. Suitably, the concentration is from about 0.05% to 1%.

[0023] Suitably, the method of the present invention includes the step of subjecting the tissue to ionizing radiation. Suitably, the dose of ionizing radiation is from about 15 to about 50 kGy.

[0024] Suitably, the method of the present invention takes from 3 days to 14 days, suitably 3 days to 12 days, suitably 3 days to 10 days, suitably 3 days to 7 days, suitably 5 days to 14 days, suitably 5 days to 12 days, suitably 5 days to 10 days, suitably 5 days to 7 days.

[0025] In another aspect, the present invention relates to a decellularized tissue scaffold produced by the method of the present invention.

[0026] Suitably, the decellularized tissue scaffold can be substantially free of anionic detergent (such as SDS) residues.

[0027] Suitably, the decellularized tissue scaffold can be substantially free of protease inhibitors, suitably free of protease inhibitors of animal origin.

[0028] Suitably, the acellular tissue scaffold can be substantially free of galactose-α-1,3-galactose (also known as α-gal) protein.

[0029] Suitably, the tissue scaffold can be a composite tissue scaffold. Suitably, the composite tissue scaffold can be a meniscus tissue scaffold (e.g., a porcine meniscus tissue scaffold), a dermal tissue scaffold (e.g., a porcine dermal tissue scaffold), or a tendon tissue scaffold (e.g., a porcine tendon tissue scaffold).

[0030] Unless the context otherwise requires, the considerations set forth in this disclosure should be considered to apply to the methods and scaffolds according to the present invention.

[0031] Throughout this specification and the claims, the words "comprising" and "including" and their variants mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps.

[0032] Throughout this specification and the claims, the singular forms include the plural forms unless the context otherwise requires. In particular, in the case of using an indefinite article, unless the context otherwise requires, this specification should be understood to cover both the plural and the singular.

[0033] Features, integers, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the present invention should be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0034] The various aspects of the present invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In the following, embodiments of the present invention will be further described with reference to the drawings, wherein:

[0036] Figure 1 An image of an acellular tissue scaffold produced by the method of the present invention is shown. It can be seen that the tissue structure of the material is retained. A is an acellular meniscus scaffold. B is an acellular dermal scaffold. C is Figure 1 Another view of the acellular dermal scaffold of B. DETAILED DESCRIPTION

[0037] The present invention is based on a new method for preparing a biological scaffold developed by the inventors, which has several advantages over existing methods.

[0038] Specifically, the inventors have found that by including at least one optimized step of incubating with a hypertonic solution and incubating with a hypotonic solution, and / or at least one optimized step of freezing / thawing the tissue, the concentration of anionic detergent required to obtain an acellular tissue scaffold can be significantly reduced.

[0039] Anionic detergents, such as SDS, are highly cytotoxic and leave processing residues in tissue scaffolds, which may be cytotoxic and thus have a negative impact on the biocompatibility of the scaffolds, even after the SDS has been washed off. The inventors have found that the concentration of the anionic detergent (such as SDS) used can be reduced such that the tissue scaffolds can be substantially free of processing residues, thereby improving the biocompatibility of the scaffolds by modifying and optimizing other steps in the decellularization process.

[0040] Another advantage of the method of the present invention is that there is no longer a need to use protease inhibitors commonly used in the art. This is beneficial because many methods use protease inhibitors of animal origin, such as aprotinin, which is derived from cattle and is associated with the risk of zoonotic disease transmission. The inventors have found that adding a rinsing step after cell lysis can sufficiently remove the proteases present in the decellularized tissue, and in addition, reducing the processing time during each step of cell lysis can reduce the impact of the proteases released from the cells. This means that there is no longer a need to use protease inhibitors. While reducing the risk of zoonotic disease transmission, this also improves the efficiency of the process.

[0041] In addition, it has also been found that the method of the present invention significantly reduces the amount of galactose-α-1,3-galactose (also known as α-gal) protein present in the decellularized tissue. This is advantageous because the α-gal protein may cause an immune response in a subject. In prior art methods, removing α-gal typically requires an additional step of treating the tissue with α-galactosidase, which degrades α-gal. The inventors have found that in the present method, this step does not have to be carried out in sequence to remove substantially all of the α-gal from the scaffold. The method of the present invention renders the scaffold substantially free of α-gal.

[0042] Method steps

[0043] The present invention relates to a method for producing a decellularized tissue scaffold, the method comprising the steps of:

[0044] · incubating the starting tissue with a degreasing agent;

[0045] · incubating the tissue with an anionic detergent;

[0046] · incubating the tissue with a hypotonic buffer at least once, incubating with a hypertonic buffer at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0047] · incubating the tissue with a DNA removing agent to obtain a decellularized tissue scaffold.

[0048] As described elsewhere in this specification, the steps of the method need not be carried out in the order provided above.

[0049] Suitably, the method may include, in some cases, the step of incubating the tissue with a protease inhibitor. Suitably a protease inhibitor of non-animal origin.

[0050] Thus, the step of incubating the tissue with a defatting agent may be carried out before the step of incubating the tissue with an anionic detergent, the step of incubating the tissue with a hypotonic buffer at least once and with a hypertonic buffer at least once, and / or the step of subjecting the tissue to at least one freeze / thaw cycle, and the step of incubating the tissue with a protease inhibitor and the step of incubating the tissue with a DNA removing agent.

[0051] Suitably, the step of incubating the tissue with an anionic agent may be carried out after the step of incubating the tissue with a defatting agent.

[0052] Suitably, the step of incubating the tissue with an anionic reagent may be carried out before the step of incubating the tissue with a DNA removing agent.

[0053] Suitably, the step of incubating the tissue with a DNA removing agent may be carried out after the step of incubating with an anionic reagent.

[0054] Those skilled in the art will know of other variations in the order of the steps of the method. Two or more steps of the invention may be combined into one step.

[0055] Suitably, the step of incubating the tissue with an anionic detergent may be combined with the step of incubating the tissue with a hypotonic buffer.

[0056] Other ways in which two or more steps of the method of the invention may be combined will be apparent to those skilled in the art.

[0057] Tissue scaffold

[0058] As used herein, the term "tissue scaffold" refers to a biological structural scaffold designed to mimic the natural extracellular matrix. Suitably, the tissue scaffold may promote the attachment, migration, proliferation and / or three-dimensional organization of cells growing therein.

[0059] A decellularized tissue scaffold can be prepared from tissue obtained from a human or animal subject or a human or animal cadaver by the method of the invention. In the context of the present disclosure, the tissue obtained from the human or animal subject for use in manufacturing a decellularized tissue scaffold may be referred to as "starting tissue" or "tissue".

[0060] Suitably, the starting tissue can be selected from the group consisting of skin (or a part of skin, such as dermis), meniscus, tendon, ligament, cartilage, muscle, blood vessel, and organ (such as heart, kidney, liver, etc.). Suitably, the starting tissue can be skin or meniscus or tendon. By way of example only, the starting tissue can be dermis, such as porcine dermis, or the starting tissue can be porcine meniscus or porcine tendon.

[0061] Suitably, the animal from which the starting tissue is obtained can be, for example, pig, non-human primate, bovine, sheep, or horse.

[0062] In the context of the present disclosure, the term "decellularized" means that the scaffold is substantially free of cellular components (e.g., cell membranes, nucleic acids, lipids, lipoid substances, and cytoplasmic components). However, it should be understood that the decellularized scaffold will retain the extracellular matrix (ECM).

[0063] As used herein, the phrase "extracellular matrix (ECM)" refers to a network of substances produced and secreted by tissue cells into the surrounding extracellular space and / or matrix, which usually endows the tissue with its mechanical and structural properties together with the tissue cells. Generally, ECM includes fibrous elements (especially collagen, elastin, or reticulin), cell adhesion polypeptides (such as fibronectin, laminin, and adhesion glycoproteins), and space-filling molecules (such as glycosaminoglycans (GAGs), proteoglycans).

[0064] "Substantially free of" cellular material means that the scaffold is substantially free of cellular components and / or substantially free of nucleic acids.

[0065] By microscopic examination using hematoxylin and eosin staining, it can be determined that the scaffold is substantially free of cellular components. Suitably, the scaffold is at least 80% free of the cellular components naturally present in the starting tissue from which the scaffold is produced. Suitably, the scaffold can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more free of cellular components.

[0066] By measuring the amount of remaining nucleic acids in the processed scaffold, it can be determined that the scaffold is substantially free of cellular material. Suitably, the scaffold is also substantially free of nucleic acids as defined below. Suitably, if the DNA amount is less than about 50 ng / mg, the DNA can be sufficiently removed from the scaffold. Suitably, if the DNA is less than about 50 ng / mg, the scaffold is substantially free of cellular material. Suitably, the DNA amount is less than about 50 ng / mg of the dry weight of the scaffold.

[0067] Accordingly, suitably, the scaffold is substantially free of cellular material in any of the following cases: (i) at least 80% thereof is free of the cellular components as defined above, or (ii) the DNA content is less than about 50 ng / mg.

[0068] Suitably, the scaffold is substantially free of cellular material when: (i) at least 80% thereof is free of the cellular components as defined above, and (ii) the DNA content is less than about 50 ng / mg.

[0069] The degree of decellularization can be determined by histochemical methods, for example, by staining the tissue with hematoxylin and eosin using standard techniques, which visualizes cell membranes and cell nuclei. Immunohistochemical staining is another method by which the degree of decellularization can be determined. Immunohistochemical staining can visualize cell-specific markers such as smooth muscle actin and histocompatibility antigens (the absence of such markers indicates decellularization). Another method by which the degree of decellularization can be determined is DNA quantification. A DNA amount equal to or less than 50 n / mg can be used as an indicator of decellularization. Other methods suitable for evaluating decellularization will be known to those skilled in the art.

[0070] Suitably, the decellularized tissue scaffold can be substantially free of α-gal. In this context, "substantially free of" means that the amount of α-gal in the scaffold is within a clinically acceptable level. Suitably, the scaffold can comprise 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of α-gal compared to the amount of α-gal naturally present in the starting tissue from which the scaffold is produced. Methods for determining the amount of α-gal will be known to those skilled in the art. By way of example only, α-gal can be measured by ELISA using a commercially available kit (such as a kit available from MyBioSource).

[0071] Degreasing agent

[0072] In the method of the present invention, the starting tissue is incubated with a defatting agent. As used herein, the term "defatting agent" refers to an agent that removes lipids and / or lipid-like substances from the starting tissue such that the starting tissue is substantially free of lipids and / or lipid-like substances. Lipids and / or lipid-like substances can include, but are not limited to, complex lipids, simple lipids, triglycerides, fatty acids, glycerophospholipids (phospholipids), pure fats such as fatty acid esters, glycerol, cerebrosides, waxes, and sterols such as cholesterol and ergosterol. Lipids and / or lipid-like substances can be derived from the starting tissue itself.

[0073] In the context of a degreasing agent, "substantially free" means that the tissue is at least 80% free of lipids and / or lipid-like substances present on the tissue prior to incubation with the degreasing agent. Suitably, the tissue can be at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or more free of lipids and / or lipid-like substances compared to the starting tissue.

[0074] Lipids and / or lipid-like substances may be insoluble in water and thus affect subsequent water-based treatments such as washing. Suitably, lipids and / or lipid-like substances can be soluble in a polar solvent. Accordingly, the degreasing agent can be a polar solvent. As used herein, the term "polar solvent" refers to a solvent that interacts with other compounds through acid-base interactions, hydrogen bonding, dipole-dipole interactions, and / or through dipole-induced dipole forces.

[0075] Suitably, the polar solvent can be selected from acetone, isopropyl alcohol, methanol, ethanol, ethyl acetate, or a mixture of two or more thereof. More suitably, the polar solvent can be acetone.

[0076] Suitably, the concentration of the polar solvent can be from about 80% to about 100% v / v. In one embodiment, the concentration of the polar solvent is about 99% v / v.

[0077] It should be understood that in the method of the present invention, the tissue can be incubated with the degreasing agent for a sufficient period of time to remove lipids and / or lipid-like substances from the tissue. Suitably, until the desired lipids and / or lipid-like substances are removed, rendering the tissue substantially free of said lipids and / or lipid-like substances.

[0078] Suitably, the incubation period with the degreasing agent can be from about 10 minutes to about 24 hours, from about 1 hour to about 5 hours, suitably from about 2 hours to about 3 hours.

[0079] Suitably, the incubation period with the degreasing agent can be about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, about 24 hours or longer. Suitably, the incubation period can be about 2 to 3 hours.

[0080] In one embodiment, the degreasing agent is acetone at a concentration of about 99% v / v. In such an embodiment, the incubation period is about 2 - 3 hours.

[0081] In one embodiment, the incubation period is about 2 hours.

[0082] In one embodiment, the incubation period is about 3 hours.

[0083] In one embodiment, there may be more than one step of incubating the tissue with a degreasing agent. In one embodiment, there may be more than one incubation period with the degreasing agent. In some embodiments, there may be two incubation periods with the degreasing agent. Suitably, each incubation period may be of a different total length of time and may include different time periods.

[0084] In some embodiments, the method comprises the steps of:

[0085] · Incubating the starting tissue with a degreasing agent for about 2 - 3 hours, wherein the degreasing agent is 99% (v / v) acetone;

[0086] · Incubating the tissue with an anionic detergent;

[0087] · Incubating the tissue with a hypotonic buffer at least once, with a hypertonic buffer at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0088] · Incubating the tissue with a DNA removing agent to obtain a decellularized tissue scaffold.

[0089] As used herein, the terms “incubation” or “incubating” refer to contacting the tissue with a relevant reagent (the relevant reagent being a degreasing agent, an anionic detergent, a hypotonic solution, a hypertonic solution, a non - animal - derived protease inhibitor, a DNA removing agent, or an antimicrobial agent). In the context of the present disclosure, the incubation period refers to the total amount of time that the tissue is in contact with the relevant reagent (i.e., incubated therewith).

[0090] The incubation period may be a single continuous time period equal to the total incubation period, or multiple time periods that together equal the total incubation period. By way of example only, in the context of incubation with acetone, the incubation period may include a single continuous time period, or for example three time periods.

[0091] Those skilled in the art will recognize that there may be rest periods between the time periods. Suitably, the tissue may rest between the time periods. Suitably, the relevant solution may be replaced between one or more of the time periods, such that the tissue is incubated with fresh relevant solution. For example, the incubation period with the degreasing agent may be 2 hours, comprising three 40 - minute time periods, wherein the degreasing agent is replaced between each time period.

[0092] In one embodiment, the incubation period with the degreasing agent is 2 hours, comprising three 40 - minute time periods.

[0093] In one embodiment, the incubation period with the degreasing agent is 3 hours, including three 1-hour time periods.

[0094] In one embodiment, the incubation period with the degreasing agent is 33 hours, including two 1-hour time periods and one 31-hour extended time period.

[0095] In some embodiments, the method comprises the following steps:

[0096] · Incubate the starting tissue with a degreasing agent for about 3 hours, wherein the degreasing agent is 99% (v / v) acetone, and wherein the incubation includes three 1-hour time periods;

[0097] · Incubate the tissue with an anionic detergent;

[0098] · Incubate the tissue with a hypotonic buffer at least once, with a hypertonic buffer at least once, and / or subject the tissue to at least one freeze / thaw cycle; and

[0099] · Incubate the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0100] In some embodiments, the method comprises the following steps:

[0101] · Incubate the starting tissue with a degreasing agent for about 2 hours, wherein the degreasing agent is 99% (v / v) acetone, and wherein the incubation includes three 40-minute time periods;

[0102] · Incubate the tissue with an anionic detergent;

[0103] · Incubate the tissue with a hypotonic buffer at least once, with a hypertonic buffer at least once, and / or subject the tissue to at least one freeze / thaw cycle; and

[0104] · Incubate the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0105] In one embodiment, the method comprises only one step of incubating the starting tissue with a degreasing agent. In one embodiment, this step lasts about 2 hours, wherein the degreasing agent is 99% (v / v) acetone, and wherein the incubation includes three 40-minute time periods. In another embodiment, the method comprises two steps of incubating the starting tissue with a degreasing agent. In one embodiment, the first step can last about 33 hours and the second step can last about 3 hours, wherein the first step includes two 1-hour time periods and one 31-hour time period, and wherein the second step includes three 1-hour time periods.

[0106] Anionic detergent

[0107] The method of the present invention includes the step of incubating the tissue in an anionic detergent.

[0108] Suitably, the incubation with the anionic detergent can be carried out before or after the incubation with the degreaser. More suitably, this step can be carried out after the incubation with the degreaser.

[0109] As used herein, the term "anionic detergent" refers to a surfactant having a negatively charged hydrophilic end. Anionic detergents can disrupt membranes and denature proteins by breaking protein-protein interactions.

[0110] Suitably, the anionic detergent can be selected from the group consisting of sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium laurate, sodium stearate, dioctyl sodium sulfosuccinate, amphoteric sodium N-lauroylsarcosinate, and any mixture of two or more thereof. More suitably, the anionic detergent is selected from the group consisting of: SDS, sodium deoxycholate, alkylbenzene sulfonates, alkyl sulfonates, alkyl sulfonates, alkyl sulfates, fluorinated fatty acid salts, silicones, fatty alcohol sulfates, polyoxyethylene fatty alcohol ether sulfates, α-olefin sulfonates, polyoxyethylene fatty alcohol phosphate ethers, alkyl alcoholamides, alkyl sulfonamidoethanols, alkyl sulfosuccinates, amino alcohol alkylbenzene sulfonates, naphthenates, alkylphenol sulfonates, and polyoxyethylene monolaurates. In one embodiment, the anionic detergent is SDS.

[0111] Suitably, the concentration of the anionic detergent (e.g., SDS or sodium deoxycholate) can be from about 0.001% to about 0.1% (w / v), from about 0.002% to about 0.018% (w / v), from about 0.004% to about 0.016% (w / v), from about 0.006% to about 0.014% (w / v), or from about 0.008 to about 0.012% (w / v).

[0112] Suitably, the concentration of the anionic detergent (e.g., SDS or sodium deoxycholate) can be about 0.2% or less (w / v), about 0.18% or less (w / v), about 0.16% or less (w / v), about 0.14% or less (w / v), about 0.12% or less (w / v), about 0.1% or less (w / v), about 0.08% or less (w / v), about 0.06% or less (w / v), about 0.04% or less (w / v), about 0.02% or less (w / v), about 0.01% or less (w / v), about 0.008% or less (w / v), about 0.006% or less (w / v), about 0.004% or less (w / v), about 0.002% or less (w / v), or about 0.001% or less (w / v).

[0113] In one embodiment, the concentration of the anionic detergent (e.g., SDS or sodium deoxycholate) is about 0.01% (w / v).

[0114] In one embodiment, the concentration of the anionic detergent (e.g., SDS or sodium deoxycholate) is about 0.1% (w / v).

[0115] The anionic detergent can be diluted in any suitable solution to achieve the desired concentration. The solution can be a hypotonic, isotonic or hypertonic solution. More suitably, the solution can be hypotonic.

[0116] Suitably, the solution can be selected from tris buffer, tris / EDTA buffer (0.1% Tris / 0.1% EDTA), tris(hydroxymethyl)aminomethane hydrochloride (tris-HCl, 10 - 100 mM), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 - 100 mM), a diluted solution of PBS (e.g., a solution containing 0.2 g of KCl, 0.2 g of KH 2 P0 4 、8 g of NaCl, or a solution containing 2.16 g of Na 2 HP0 4 *7H 2 0 of 1000 ml H 2 O, and a diluted solution of physiological saline (e.g., containing 0.9% NaCl).

[0117] In one embodiment, the solution is a hypotonic tris buffer.

[0118] Suitably, the incubation period with the anionic detergent (e.g., SDS or sodium deoxycholate) is long enough to achieve cell lysis in the tissue. Suitably, this can be from about 10 to about 100 hours, suitably from about 20 to about 80 hours.

[0119] Suitably, the incubation period with the anionic detergent can be about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 75 hours, about 80 hours, about 85 hours, about 90 hours, about 95 hours, about 100 hours, or longer.

[0120] It should be understood that the incubation period with an anionic detergent can depend on the type of starting tissue used and / or the detergent itself. By way of example only, porcine dermis can be incubated with an anionic detergent such as SDS for about 21 or 24 hours, while porcine meniscus can be incubated with an anionic detergent such as SDS for about 80 hours, and porcine tendon can be incubated with an anionic detergent for about 48 hours. Those skilled in the art can appropriately determine the optimal incubation time with an anionic detergent by determining whether cell lysis has occurred in the tissue, which can be determined, for example, using microscopy.

[0121] In some embodiments, the method comprises the steps of:

[0122] · Incubating the starting tissue with a defatting agent for about 2 - 3 hours, wherein the defatting agent is 99% (v / v) acetone;

[0123] · Incubating the tissue with an anionic detergent, wherein the anionic detergent is 0.01% (w / v) SDS;

[0124] · Incubating the tissue with a hypotonic buffer at least once, incubating with a hypertonic buffer at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0125] · Incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0126] In one embodiment, the tissue is incubated with 0.01% SDS, an anionic detergent, for 21 or 24 hours.

[0127] In one embodiment, the tissue is incubated with 0.01% SDS, an anionic detergent, for 80 hours, including four 20 - hour periods.

[0128] In one embodiment, the method comprises two steps of incubating the tissue with 0.1% SDS, an anionic detergent. In one embodiment, each step is about 24 hours.

[0129] In some embodiments, the method comprises the steps of:

[0130] · Incubating the starting tissue with a defatting agent for about 3 hours, wherein the defatting agent is 99% (v / v) acetone, and wherein the incubation comprises three 1 - hour periods;

[0131] · Incubating the tissue with an anionic detergent for about 24 hours, wherein the anionic detergent is 0.01% (w / v) SDS;

[0132] · Incubating the tissue with a hypotonic buffer at least once, incubating with a hypertonic buffer at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0133] ·Incubate the tissue with a DNA removal agent to obtain a decellularized tissue scaffold.

[0134] In some embodiments, the method comprises the steps of:

[0135] ·Incubate the starting tissue with a defatting agent for about 2 hours, wherein the defatting agent is 99% (v / v) acetone, and wherein the incubation comprises three 40-minute periods;

[0136] ·Incubate the tissue with an anionic detergent for about 80 hours, wherein the anionic detergent is 0.01% (w / v) SDS, and wherein the incubation comprises four 20-hour periods;

[0137] ·Incubate the tissue with a hypotonic buffer at least once, with a hypertonic buffer at least once, and / or subject the tissue to at least one freeze / thaw cycle; and

[0138] ·Incubate the tissue with a DNA removal agent to obtain a decellularized tissue scaffold.

[0139] Hypotonic and hypertonic solutions

[0140] The method of the present invention comprises the steps of incubating the tissue with a hypotonic solution at least once, with a hypertonic solution at least once, and / or subjecting the tissue to at least one freeze / thaw cycle. Suitably, these steps are used to lyse the cells present in the tissue.

[0141] Suitably, incubating the tissue with a hypotonic solution at least once and incubating the tissue with a hypertonic solution at least once achieve a similar effect to subjecting the tissue to at least one freeze / thaw cycle; both of these steps are used to lyse the cells. Suitably, either step can be used, or both can be used in combination. Suitably, thus the method can comprise incubating the tissue with a hypotonic solution at least once and incubating with a hypertonic solution at least once, or suitably, the method can comprise subjecting the tissue to at least one freeze / thaw cycle. Alternatively, the method can comprise incubating the tissue with a hypotonic solution at least once and incubating with a hypertonic solution at least once, and subjecting the tissue to at least one freeze / thaw cycle.

[0142] "Freeze / thaw cycle" means freezing the tissue (e.g., placing it in liquid nitrogen, or in a -80 °C or -20 °C freezer), and then thawing it (e.g., at 2 - 8 °C or 34 °C or 37 °C or at room temperature).

[0143] Suitably, the tissue can be subjected to multiple freeze / thaw cycles. Suitably, the tissue can be subjected to one to five freeze / thaw cycles. In one embodiment, the tissue is subjected to two or three freeze / thaw cycles.

[0144] The term "hypotonic solution" refers to a solution in which the solute concentration is lower than the solute concentration within the tissue cells. The term "hypertonic solution" refers to a solution in which the solute concentration is greater than the solute concentration within the tissue cells.

[0145] By way of example only, hypotonic solutions can be selected from the group consisting of tris / EDTA buffer (0.1% Tris / 0.1% EDTA), tris(hydroxymethyl)aminomethane hydrochloride (tris-HCl, 10 - 100 mM), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 - 100 mM), a diluted solution of PBS (e.g., a solution containing 0.2 g of KCl, 0.2 g of KH 2 PO 4 、8 g of NaCl, or a solution containing 2.16 g of Na 2 HPO 4 *7H 2 O in 1000 ml of H 2 O, and a diluted solution of physiological saline (e.g., containing 0.9% NaCl). Those skilled in the art will be familiar with other hypotonic solutions.

[0146] In one embodiment, the hypotonic solution is 0.1% tris / 0.1% EDTA.

[0147] The incubation period with the hypotonic solution can be from about 30 minutes to 100 hours, suitably from 10 hours to about 100 hours, or from about 20 hours to 80 hours.

[0148] Suitably, the incubation period with the hypotonic solution can be about 30 minutes, 1 hour, 5 hours, 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, about 50 hours, about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 75 hours, about 80 hours, about 85 hours, about 90 hours, about 95 hours, about 100 hours, or longer.

[0149] It should be understood that the incubation period with the hypotonic solution can depend on the type of starting tissue used and / or the hypotonic solution itself. For example, a more hypotonic solution may require a shorter incubation period compared to a less hypotonic solution. By way of example only, porcine dermis can be incubated in a hypotonic solution for about 21 - 24 hours, while porcine meniscus can be incubated with a hypotonic solution for about 40 minutes, and porcine tendon can be incubated with a hypotonic solution for up to 24 hours, suitably 1 - 4 hours in some cases, and 24 hours in some cases. For example, a skilled person can determine the appropriate incubation time by using the above method to evaluate when the tissue is decellularized.

[0150] As mentioned elsewhere in this specification, an anionic detergent can be diluted to a desired concentration in a hypotonic solution. In such an embodiment, the step of incubating the tissue with the anionic detergent and the step of incubating the tissue with the hypotonic solution at least once can be combined into one step.

[0151] Thus, in one embodiment, the method comprises the following steps:

[0152] · Incubating the starting tissue with a degreasing agent;

[0153] · Incubating the tissue with an anionic detergent and a hypotonic solution;

[0154] · Incubating the tissue with a hypotonic buffer at least once and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0155] · Incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0156] The method of the present invention comprises the step of incubating the tissue with a hypotonic solution at least once. Suitably, the tissue can be incubated with the hypotonic solution once, twice, three times or more. In one embodiment, the tissue can be incubated with the hypotonic solution twice. In one embodiment, the tissue can be incubated with the hypotonic solution six times.

[0157] Suitably, when the tissue is incubated with the hypotonic solution twice or more, one of the incubations can be combined with the step of incubating the tissue with the anionic detergent. This can be achieved by diluting the anionic detergent to a desired concentration in the hypotonic agent. Suitably, the second incubation with the hypotonic solution can be carried out before or after the incubation with the anionic detergent.

[0158] Thus, in one embodiment, the method comprises the following steps:

[0159] · Incubating the starting tissue with a degreasing agent;

[0160] · Incubating the tissue with an anionic detergent and a first hypotonic solution;

[0161] · Incubating the tissue with a second hypotonic solution at least once, incubating with a hypertonic solution at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0162] · Incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0163] Suitably, in the case where incubation with a hypotonic solution is carried out before the step of incubation with an anionic detergent, it can be carried out before or after the step of incubation with a degreaser. More suitably, in the case where incubation with a hypotonic solution is carried out before the step of incubation with an anionic detergent, it can be carried out after the step of incubation with a degreaser. The step of incubating the tissue in a hypotonic solution can be carried out before or after subjecting the tissue to at least one freeze / thaw cycle. Suitably, the step of incubating the tissue in a hypotonic solution can be carried out before subjecting the tissue to at least one freeze / thaw cycle. After the step of incubating the tissue with a hypotonic solution, there can be a step of incubating the tissue with a hypertonic solution. Incubation with a hypertonic solution can be carried out directly after incubation with a hypotonic solution. Alternatively, incubation with a hypertonic solution can be carried out after the tissue has been subjected to at least one freeze / thaw cycle.

[0164] Suitably, in one embodiment, in the case where incubation with a hypotonic solution is carried out before the step of incubation with an anionic detergent, the method can comprise the steps of:

[0165] · incubating the starting tissue with a degreaser;

[0166] · incubating the tissue with a first hypotonic solution;

[0167] · subjecting the tissue to at least one freeze / thaw cycle;

[0168] · incubating the tissue with a first hypertonic solution;

[0169] · incubating the tissue with an anionic detergent and an optional second hypotonic solution;

[0170] · incubating the tissue with a second hypertonic solution; and

[0171] · incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0172] Suitably, in the case where incubation with a hypotonic solution is carried out after incubation with an anionic detergent, it can also be carried out after the tissue has been incubated with a degreaser and / or incubated with a hypertonic solution. In such an embodiment, the method can comprise the steps of:

[0173] · incubating the starting tissue with a degreaser;

[0174] · incubating the tissue with an anionic detergent;

[0175] · incubating the tissue with a hypertonic solution;

[0176] · incubating the tissue with a hypotonic solution; and

[0177] · incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0178] Suitably, the first and second solutions can be the same solution or different solutions. Suitably, the first and second solutions of any component of the method are selected from the components described herein. This also applies to any number of solutions of the same component. For example, third and fourth hypertonic solutions can be used. Suitably, these solutions can all be the same solution or different solutions selected from those described herein.

[0179] In one embodiment, the method can include six steps of incubating the tissue with hypotonic treatment. Suitably, the first two steps include incubating for a period of 1-4 hours with a hypotonic solution. Suitably, the third, fourth, fifth, and sixth steps include incubating for 24 hours with a hypotonic solution. Suitably, at least one of the third, fourth, fifth, and sixth steps includes an anionic detergent. Suitably, the first and second steps can each be before a freeze / thaw cycle.

[0180] In one embodiment, the method can include two steps of incubating the tissue with hypotonic treatment. Suitably, the time for the two steps is about 21 hours. Suitably, at least one step includes an anionic detergent.

[0181] As used herein, the term "hypertonic solution" refers to a solution in which the solute concentration is greater than the intracellular solute concentration.

[0182] By way of example only, hypertonic solutions can be selected from, for example, tris / sodium chloride solution (0.6% Tris / 8-9% sodium chloride solution), sugar solution, salt solution, 5% dextran (sugar) and 0.45% sodium chloride, 5% dextran and 0.9% sodium chloride salt, and aqueous solutions of 10% dextran. In one embodiment, the hypertonic solution is 0.6% Tris / 8-9% sodium chloride solution.

[0183] In one embodiment, the hypertonic solution is tris / sodium chloride solution (0.6% Tris / 8.2% sodium chloride solution).

[0184] Suitably, the incubation period with the hypertonic solution can be from about 1 hour to about 48 hours, from about 4 hours to 30 hours, from about 8 hours to about 24 hours.

[0185] Suitably, the incubation period with the hypertonic solution can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, or longer.

[0186] It should be understood that the incubation period with the hypertonic solution can depend on the type of starting tissue used and / or the hypertonic solution. By way of example only, porcine dermis can be incubated with the hypertonic solution for about 21 - 24 hours, while porcine meniscus can be incubated with the hypertonic solution for about 8 hours, and porcine tendon can be incubated with the hypertonic solution for about 18 hours. Those skilled in the art can determine the appropriate incubation time by performing routine experiments on the tissues in question.

[0187] In one embodiment, the incubation period with the hypertonic tris / sodium chloride solution (0.6% Tris / 8.2% sodium chloride solution) is 21 - 24 hours.

[0188] In one embodiment, the incubation period with the hypertonic tris / sodium chloride solution (0.6% Tris / 9% sodium chloride solution) is 8 hours. In one embodiment, the method includes two incubation periods with the hypertonic tris / sodium chloride solution (0.6% Tris / 9% sodium chloride solution), each incubation period being 8 hours.

[0189] In one embodiment, the incubation period with the hypertonic tris / sodium chloride solution (0.6% Tris / 8.2% sodium chloride solution) is about 18 hours.

[0190] Suitably, the method includes incubating the tissue with the hypertonic solution at least once. Suitably, the method includes incubating the tissue with the hypertonic solution one, two, three or more times. More suitably, the method can include incubating the tissue with the hypertonic solution one or two times. In one embodiment, the method includes incubating the tissue with the hypertonic solution once.

[0191] In an embodiment where the tissue is incubated with the hypertonic solution once, the step of incubating the tissue with the hypertonic solution can be carried out after incubating with an anionic detergent (with or without a hypotonic solution).

[0192] In an embodiment where the tissue is incubated with the hypertonic solution twice, the step of incubating the tissue with the hypertonic solution can be carried out once before incubating with the anionic detergent (with or without a hypotonic solution), and once after incubating with the anionic detergent (with or without a hypotonic solution).

[0193] In one embodiment, the method includes:

[0194] · incubating the starting tissue with a degreaser;

[0195] · incubating the tissue with an anionic detergent and a first hypotonic solution;

[0196] · incubating the tissue with the hypertonic solution;

[0197] · incubating the tissue with a second hypotonic solution; and

[0198] · Incubate the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0199] In one embodiment, the method includes:

[0200] · Incubate the starting tissue with a degreasing agent;

[0201] · Incubate the tissue with an anionic detergent and a first hypotonic solution for 24 hours, wherein the first hypotonic solution comprises a 0.1% tris / 0.1% EDTA solution;

[0202] · Incubate the tissue with a hypertonic solution for 24 hours, wherein the hypertonic solution comprises a 0.6% Tris / 8-9% sodium chloride solution;

[0203] · Incubate the tissue with a second hypotonic solution for 24 hours, wherein the second hypotonic solution comprises a 0.1% tris / 0.1% EDTA solution; and

[0204] · Incubate the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0205] In one embodiment, the method includes:

[0206] · Incubate the starting tissue with a degreasing agent;

[0207] · Incubate the tissue with a first hypertonic solution;

[0208] · Incubate the tissue with an anionic detergent and a hypotonic solution;

[0209] · Incubate the tissue with a second hypertonic solution; and

[0210] · Incubate the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0211] In one embodiment, the method includes:

[0212] · Incubate the starting tissue with a degreasing agent;

[0213] · Incubate the tissue with a first hypertonic solution for up to 8 hours, wherein the first hypertonic solution comprises a 0.6% tris / 9% sodium chloride solution;

[0214] · Incubate the tissue with an anionic detergent and a hypotonic solution for 4 twenty-hour periods, wherein the hypotonic solution comprises a 0.1% Tris / 0.1% EDTA solution;

[0215] · Incubate the tissue with a second hypertonic solution for up to 8 hours, wherein the second hypertonic solution comprises a 0.6% Tris / 8-9% sodium chloride solution; and

[0216] · Incubate the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0217] In one embodiment, the method comprises:

[0218] · incubating the starting tissue with a degreasing agent;

[0219] · incubating the tissue with a first hypotonic solution;

[0220] · subjecting the tissue to one or more freeze / thaw cycles;

[0221] · incubating the tissue with a first hypertonic solution;

[0222] · incubating the tissue with an anionic detergent and a second hypotonic solution;

[0223] · incubating the tissue with a second hypertonic solution; and

[0224] · incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0225] In one embodiment, the method comprises:

[0226] · incubating the starting tissue with a degreasing agent;

[0227] · incubating the tissue with a first hypotonic solution, wherein the first hypotonic solution comprises a 0.1% Tris / 0.1% EDTA solution;

[0228] · subjecting the tissue to three freeze / thaw cycles;

[0229] · incubating the tissue with a first hypertonic solution for up to 8 hours, wherein the first hypertonic solution comprises a 0.6% tris / 8-9% sodium chloride solution;

[0230] · incubating the tissue with an anionic detergent and a second hypotonic solution for four 20-hour periods, wherein the second hypotonic solution comprises a 0.1% Tris / 0.1% EDTA solution;

[0231] · incubating the tissue with a second hypertonic solution for up to 8 hours, wherein the second hypertonic solution comprises a 0.6% Tris / 8-9% sodium chloride solution; and

[0232] · incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0233] Protease inhibitor

[0234] Suitably, the method of the present invention does not include protease inhibitors, especially protease inhibitors of animal origin. Thus, suitably, the method does not include the step of incubating the tissue with a protease inhibitor.

[0235] In some embodiments, the method comprises the following steps:

[0236] · Incubate the starting tissue with a degreasing agent;

[0237] · Incubate the tissue with an anionic detergent;

[0238] · Incubate the tissue with a hypotonic solution at least once, with a hypertonic solution at least once, and / or subject the tissue to at least one freeze / thaw cycle; and

[0239] · Incubate the tissue with a DNA removing agent to obtain a decellularized tissue scaffold,

[0240] wherein the method does not include protease inhibitors of animal origin.

[0241] However, although not necessarily required for the method of the present invention, the method may include the step of incubating the tissue with a protease inhibitor of non - animal origin. As used herein, the term "protease inhibitor" refers to an agent that inhibits the proteolytic activity of a protease. The term "non - animal origin" means that the protease inhibitor is not an inhibitor obtained from an animal source and does not occur naturally in an animal.

[0242] Suitably, the protease inhibitor of non - animal origin can be a metalloprotease inhibitor. Suitably, it is selected from any chelating agent. Suitably, the chelating agent acts as a protease inhibitor and prevents the decomposition of the tissue. Suitably, the protease inhibitor of non - animal origin is selected from, for example, EDTA, NTA, ATMP, EDTMP and HEDP. In one embodiment, the protease inhibitor of non - animal origin is EDTA.

[0243] Suitably, the protease inhibitor of non - animal origin is used at a concentration of about 0.01% to about 1%, about 0.05% to about 0.5%, or about 0.75% to about 0.25%.

[0244] Suitably, the protease inhibitor of non - animal origin is used at a concentration of about 1%, about 0.75%, about 0.5%, about 0.25%, about 0.1%, about 0.05% or about 0.01%. In one embodiment, the protease inhibitor of non - animal origin is used at a concentration of about 0.1%.

[0245] In a suitable embodiment, the protease inhibitor of non - animal origin is EDTA at a concentration of about 0.1%.

[0246] It should be understood that the step of incubating the tissue with a protease inhibitor of non - animal origin can be carried out before or after the step of incubating the tissue with a DNA removing agent.

[0247] Suitably, the step of incubating the tissue with a protease inhibitor of non-animal origin can be combined with the step of incubating the tissue with a hypotonic agent.

[0248] Suitably, the step of incubating the tissue with a hypotonic solution can be combined with the step of incubating the tissue with a protease inhibitor of non-animal origin. Suitably, the combined incubation of the tissue in the hypotonic solution and the protease inhibitor of non-animal origin is carried out after incubation with a degreasing agent, an anionic detergent (with or without a hypotonic agent), and a hypertonic solution.

[0249] In some embodiments, the method comprises the steps of:

[0250] · Incubating the starting tissue with a degreasing agent for about 2 - 3 hours, wherein the degreasing agent is 99% (v / v) acetone;

[0251] · Incubating the tissue with an anionic detergent, wherein the anionic detergent is 0.01% (w / v) SDS;

[0252] · Incubating the tissue with a hypotonic buffer at least once, incubating the tissue with a hypertonic buffer at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0253] · Incubating the tissue with a protease inhibitor of non-animal origin, wherein the protease inhibitor is 0.1% EDTA, and incubating the tissue with a DNA remover to obtain a decellularized tissue scaffold.

[0254] Suitably, the protease inhibitor of non-animal origin can be diluted to a desired concentration in any suitable solution. By way of example only, the protease inhibitor of non-animal origin (such as EDTA) can be diluted in phosphate buffered saline or in a hypotonic buffer solution (0.1% tris).

[0255] It should be understood that when the protease inhibitor of non-animal origin (such as EDTA) is diluted in a hypotonic solution, the step of incubating the tissue with the hypotonic solution and the protease inhibitor of non-animal origin can be combined into one step. By way of example, in such an embodiment, the method of the present invention comprises the steps of:

[0256] · Incubating the starting tissue with a degreasing agent;

[0257] · Incubating the tissue with an anionic detergent;

[0258] · Incubating the tissue with a hypertonic solution;

[0259] · Incubating the tissue with a hypotonic solution; and

[0260] · Incubating the tissue with a protease inhibitor of non-animal origin and a second hypotonic solution, wherein the protease inhibitor of non-animal origin is EDTA; and

[0261] · Incubate the tissue with a DNA removing agent to obtain a decellularized tissue scaffold.

[0262] In one embodiment, when the protease inhibitor of non-animal origin is not diluted in a hypotonic solution, the method may comprise the following steps:

[0263] · Incubate the starting tissue with a degreasing agent;

[0264] · Incubate the tissue with a first hypotonic solution;

[0265] · Subject the tissue to at least one freeze / thaw cycle;

[0266] · Incubate the tissue with a first hypertonic solution;

[0267] · Incubate the tissue with an anionic detergent;

[0268] · Incubate the tissue with a second hypertonic solution;

[0269] · Incubate the tissue with a DNA removing agent; and

[0270] · Incubate the tissue with a protease inhibitor of non-animal origin, wherein the protease inhibitor of non-animal origin is EDTA; to obtain a decellularized tissue scaffold.

[0271] Optionally, the anionic detergent may further comprise a hypotonic solution.

[0272] Suitably, the incubation period with the protease inhibitor of non-animal origin (such as EDTA) is sufficient to inhibit proteases in the tissue, suitably, this may be about 0.5 hours to 48 hours, or about 1 hour to about 24 hours.

[0273] Suitably, the incubation period with the protease inhibitor of non-animal origin may be about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 20 hours, about 22 hours, about 24 hours, about 28 hours, about 30 hours, about 36 hours, about 42 hours, or 48 hours, or longer.

[0274] It should be understood that the incubation period with the protease inhibitor of non-animal origin may depend on the type of starting tissue used and / or the inhibitor itself. By way of example only, porcine dermis may be incubated with an inhibitor such as EDTA for about 24 hours, while porcine meniscus may be incubated with an inhibitor such as EDTA for about 3 hours. Those skilled in the art can determine the appropriate incubation time by performing routine experiments on the tissues discussed.

[0275] In one embodiment, the incubation period with a protease inhibitor of non-animal origin, 0.1% EDTA, is 24 hours.

[0276] In one embodiment, the incubation period with a protease inhibitor of non-animal origin, 0.1% EDTA, is 3 hours.

[0277] The inventors unexpectedly found that the method of the present invention can produce acellular tissue scaffolds even without the use of protease inhibitors (suitably, even when using only protease inhibitors of non-animal origin). Aprotinin is a protease inhibitor, particularly bovine pancreatic trypsin inhibitor. Since it is derived from cattle, it may transmit zoonotic diseases. Suitably, the method does not use or does not contain protease inhibitors of animal origin. Suitably, the method does not use or does not contain aprotinin.

[0278] Suitably, the method of the present invention may further include one or more rinsing steps to help remove proteases released from lysed cells within the tissue.

[0279] Accordingly, suitably, the rinsing step may be carried out after incubating the tissue with a hypotonic solution, after incubating the tissue with a hypertonic solution, and / or after subjecting the tissue to a freeze / thaw cycle.

[0280] Suitably, the method includes a rinsing step after each step of cell lysis.

[0281] Accordingly, suitably, the rinsing step is carried out after incubating the tissue with a hypotonic solution, and after incubating the tissue with a hypertonic solution, and after subjecting the tissue to a freeze / thaw cycle. Accordingly, suitably, the method may include multiple rinsing steps. Suitably, each rinsing step may include one or more rinses of the tissue. Suitably, each rinsing step includes at least three rinses of the tissue.

[0282] In some embodiments, the method includes the following steps:

[0283] · Incubating the starting tissue with a degreasing agent;

[0284] · Incubating the tissue with an anionic detergent;

[0285] · Incubating the tissue with a hypotonic solution at least once, with a hypertonic solution at least once, and / or subjecting the tissue to at least one freeze / thaw cycle;

[0286] · Rinsing the tissue; and

[0287] · Incubating the tissue with a DNA remover to obtain an acellular tissue scaffold.

[0288] In some embodiments, the method includes the following steps:

[0289] · Incubate the starting tissue with a degreasing agent;

[0290] · Incubate the tissue with an anionic detergent;

[0291] · Incubate the tissue with a hypotonic solution;

[0292] · Rinse the tissue;

[0293] · Incubate the tissue with a hypertonic solution;

[0294] · Rinse the tissue; and

[0295] · Incubate the tissue with a DNA removal agent to obtain a decellularized tissue scaffold.

[0296] In some embodiments, the method comprises the steps of:

[0297] · Incubate the starting tissue with a degreasing agent;

[0298] · Incubate the tissue with an anionic detergent;

[0299] · Incubate the tissue with a hypotonic solution;

[0300] · Rinse the tissue;

[0301] · Incubate the tissue with a hypertonic solution;

[0302] · Rinse the tissue;

[0303] · Subject the tissue to at least one freeze / thaw cycle;

[0304] · Rinse the tissue; and

[0305] · Incubate the tissue with a DNA removal agent to obtain a decellularized tissue scaffold.

[0306] Suitably, rinsing the tissue comprises rinsing the tissue with a rinsing solution. Suitably, the rinsing solution is an isotonic solution. Suitably, the rinsing solution is a saline solution.

[0307] Suitably, in each embodiment including such one or more rinsing steps, the method does not include protease inhibitors.

[0308] In some embodiments, the method may include the step of incubating the tissue with a protease or proteinase enzyme. Suitably it is a protease or proteinase enzyme of animal origin. In one embodiment, trypsin is used, suitably porcine-derived trypsin. In one embodiment, the tissue is incubated with a protease or proteinase enzyme for a period of about 72 hours.

[0309] DNA remover

[0310] The method of the present invention includes the step of incubating the tissue with a DNA remover. As used herein, the term "DNA remover" refers to a reagent that denatures and / or cleaves DNA.

[0311] Suitably, the DNA remover can be selected from, for example, endonucleases, acids or bases.

[0312] Suitably, the endonuclease can be a human endonuclease. Suitably, the endonuclease can be selected from DNase type I, DNase type II, DNase III, RNase, or any combination of two or more thereof. Alternatively, the endonuclease can be a bacterially-derived endonuclease, or a combination of human- and bacterially-derived endonucleases, any combination of two or more thereof. Suitably, the endonuclease can be DNase type I. Suitably, the endonuclease can be a bacterially-derived endonuclease, such as a genetically engineered Serratia marcessens endonuclease, which can be selected from any commercially available endonuclease, such as Benzonase®.

[0313] Suitably, the concentration range of the endonuclease can be from about 0.5 to 50 U / ml. In one embodiment, the concentration of the endonuclease is about 5 U / ml.

[0314] Suitably, the endonuclease can be present in a reaction buffer. Suitably, the reaction buffer can include Tris and magnesium chloride hexahydrate. Suitably, the reaction buffer can include 0.6% Tris and 0.2% magnesium chloride hexahydrate.

[0315] In some embodiments, the method includes the following steps:

[0316] · Incubating the starting tissue with a defatting agent for about 2 - 3 hours, wherein the defatting agent is 99% (v / v) acetone;

[0317] · Incubating the tissue with an anionic detergent, wherein the anionic detergent is 0.01% (w / v) SDS;

[0318] · Incubating the tissue with a hypotonic buffer at least once, incubating the tissue with a hypertonic buffer at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and

[0319] · Incubating the tissue with a DNA remover, wherein the DNA remover is DNase type I at 5 U / ml, thereby obtaining a decellularized tissue scaffold.

[0320] Suitably, the incubation period with the DNA remover is long enough to reduce the DNA to a level below 50 ng / mg. Suitably, this can be from about 2 to about 24 hours, suitably from about 2 to about 12 hours. Suitably, the incubation period with the DNA remover can be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, or longer.

[0321] It should be understood that the incubation period with the DNA remover can depend on the type of starting tissue used and / or the reagent itself. By way of example only, porcine dermis can be incubated with a DNA remover such as endonuclease for about 3 hours, while porcine meniscus can be incubated with a DNA remover such as endonuclease for about 22 hours, and porcine tendon can be incubated for about 6 hours. If the amount of DNA is less than about 50 ng / mg, DNA can be sufficiently removed from the tissue. Those skilled in the art will understand that the removal of DNA from tissue can be optimized by routine experimentation involving different concentrations and / or incubation times with the DNA remover.

[0322] In one embodiment, the incubation period with an endonuclease at 5 U / ml of DNA remover is 3 hours.

[0323] In one embodiment, the incubation period with an endonuclease at 5 U / ml of DNA remover is 22 hours, including two 2-hour periods and one 18-hour period.

[0324] In one embodiment, the incubation period with an endonuclease at 5 U / ml of DNA remover is 6 hours, including three 2-hour periods.

[0325] In one embodiment, the DNA remover is Benzonase®.

[0326] The step of incubating the tissue with the DNA remover can be carried out before or after the step of incubating the tissue with a protease inhibitor of non-animal origin, if present.

[0327] Antimicrobial agent

[0328] The method of the present invention can also include the step of incubating the tissue in an antimicrobial agent. This step may be particularly beneficial in the context of a method for producing a decellularized tissue scaffold from a non-human starting tissue, as such tissue is associated with a risk of transmitting zoonotic diseases.

[0329] As used herein, the term "antimicrobial agent" refers to any natural, synthetic, or semi-synthetic compound having antibacterial, antifungal, antiviral, and / or antiparasitic activity. In the context of the present disclosure, such activity can include reducing the number of live bacteria, fungi, viruses, and / or parasites in the starting tissue and / or tissue scaffold, and / or reducing the chance of contamination and subsequent infection of the recipient of the tissue scaffold when used in vivo. Reducing the number of live microorganisms can be achieved by restricting, preventing, and / or inhibiting the growth of microorganisms and / or killing the microorganisms.

[0330] Suitably, the antimicrobial agent can be an oxidizing agent. As used herein, the term "oxidizing agent" refers to a reagent capable of oxidizing the microbial cell membrane, resulting in lysis and death of the microbial cell.

[0331] Suitably, the oxidizing agent can be peracetic acid (PAA). Suitably, the concentration of PAA can be from about 500 to about 10,000 ppm. Suitably, the concentration of PAA can be from about 500 ppm to 1500 ppm. In one embodiment, the concentration of PAA is 1000 ppm, which can also be written as 0.1% in other ways.

[0332] Suitably, the oxidizing agent (e.g., PAA) can have a pH of about 6 to about 8. More suitably, the oxidizing agent (e.g., PAA) can have a pH of about 7.

[0333] Suitably, the oxidizing agent can be present in a buffer, suitably for example PBS buffer.

[0334] It should be understood that the antimicrobial agent can include a single antimicrobial agent or two or more antimicrobial agents. For example, the antimicrobial agent can include two antimicrobial agents.

[0335] In embodiments where the antimicrobial agent includes two or more antimicrobial agents, at least one reagent can be a mild oxidizing agent (e.g., PAA). Suitably, in embodiments where the antimicrobial agent includes two or more antimicrobial agents, at least one reagent can be a mild oxidizing agent (e.g., PAA), and at least one reagent can be an antibiotic.

[0336] Suitably, the antibiotic can be selected from the group consisting of penicillin-streptomycin, streptomycin, ampicillin, actinomycin D, carbenicillin, cefotaxime, fosfomycin, gentamicin, kanamycin, neomycin, and polymyxin B.

[0337] In the method of the present invention, the tissue can be incubated with an antimicrobial agent (such as a mild oxidizing agent, such as PAA) for a sufficient time until the tissue is substantially free of live microorganisms. Suitably, the incubation can be for about 15 minutes to about 24 hours, suitably for about 30 minutes to about 12 hours, suitably for about 1 hour to about 6 hours, suitably for about 2 hours to about 6 hours.

[0338] More suitably, the incubation with the antimicrobial agent (such as an oxidizing agent, such as PAA) can last for about 1 hour. In this context, "substantially free of" means that the tissue is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more free of the microorganisms present in the tissue before incubation with the antimicrobial agent. A person skilled in the art can determine the appropriate incubation time for removing microorganisms from the tissue by, for example, routine experiments.

[0339] In one embodiment, the incubation with the antimicrobial agent of 0.1% PAA is for 1 hour or about 3 hours.

[0340] The step of incubating with the antimicrobial agent can be carried out at least once, twice, three times or more. Suitably, the step of incubating with the antimicrobial agent can be carried out once or twice. By way of example only, the incubation with the antimicrobial agent (such as an oxidizing agent, such as PAA) can be carried out before incubation with the degreasing agent and / or after incubation with the protease inhibitor or DNA remover. Suitably, it can be directly incubated with PAA before packaging the acellular tissue scaffold. In addition, further incubation with the antimicrobial agent can be carried out after the step of subjecting the tissue to at least one freeze / thaw cycle.

[0341] In some embodiments, the method comprises the following steps:

[0342] · Optionally incubating the starting tissue with an antimicrobial agent;

[0343] · Incubating the tissue with a degreasing agent;

[0344] · Incubating the tissue with an anionic detergent;

[0345] · Incubating the tissue with a hypotonic buffer at least once, incubating with a hypertonic buffer at least once, and / or subjecting the tissue to at least one freeze / thaw cycle;

[0346] · Incubating the tissue with a DNA remover; and

[0347] · Incubating the tissue with an antimicrobial agent to obtain an acellular tissue scaffold.

[0348] In some embodiments, the method comprises the following steps:

[0349] · Incubate the starting tissue with a defatting agent for about 2 - 3 hours, where the defatting agent is 99% (v / v) acetone;

[0350] · Incubate the tissue with an anionic detergent, where the anionic detergent is 0.01% (w / v) SDS;

[0351] · Incubate the tissue with a hypotonic buffer at least once, with a hypertonic buffer at least once, and / or subject the tissue to at least one freeze / thaw cycle;

[0352] · Incubate the tissue with a DNA removal agent, where the DNA removal agent is DNase type I at 5 U / ml; and

[0353] · Incubate the tissue with an antimicrobial agent, where the antimicrobial agent is 0.1% PAA, thereby obtaining a decellularized tissue scaffold.

[0354] In one embodiment, the method may include two steps of incubation with an antimicrobial agent. Suitably, where the antimicrobial agent is 0.1% PAA. Suitably, the first step includes an incubation period of about 1 hour with the antimicrobial agent. Suitably, the second step includes an incubation period of about 3 hours with the antimicrobial agent.

[0355] Ionizing radiation

[0356] Animal tissues typically must be exposed to a disinfection / sterilization process that has been shown to reduce zoonoses by 6 - log. However, this level of reduction generally cannot be achieved by traditional sterilization methods, such as gamma irradiation at a dose of 25 kGy. The present inventors unexpectedly found that such a sterilization level can be achieved by using ionizing radiation. Accordingly, the method of the present invention may further include the step of subjecting the decellularized biological scaffold to ionizing radiation. Suitably, the ionizing radiation can be from about 15 kGy to about 50 kGy. Suitably, the ionizing radiation can be from about 18 kGy to 25 kGy, and suitably, the ionizing radiation can be from 25 kGy to 27.5 kGy.

[0357] Suitably, the ionizing radiation can be carried out before or after packaging the tissue. Suitably, the ionizing radiation can be carried out after packaging the tissue.

[0358] Suitably, the ionizing radiation can be carried out with or without dry ice.

[0359] In some embodiments, the method includes the following steps:

[0360] · Incubate the starting tissue with a defatting agent;

[0361] · Incubate the tissue with an anionic detergent;

[0362] · Incubate the tissue with a hypotonic buffer at least once, with a hypertonic buffer at least once, and / or subject the tissue to at least one freeze / thaw cycle;

[0363] · Incubate the tissue with a DNA remover;

[0364] · Incubate the tissue with an antimicrobial agent to obtain a decellularized tissue scaffold;

[0365] · Optionally package the scaffold; and

[0366] · Subject the scaffold to ionizing radiation.

[0367] In some embodiments, the method comprises the steps of:

[0368] · Incubate the starting tissue with a degreaser for about 2 - 3 hours, wherein the degreaser is 99% (v / v) acetone;

[0369] · Incubate the tissue with an anionic detergent, wherein the anionic detergent is 0.01% (w / v) SDS;

[0370] · Incubate the tissue with a hypotonic buffer at least once, with a hypertonic buffer at least once, and / or subject the tissue to at least one freeze / thaw cycle;

[0371] · Incubate the tissue with a DNA remover, wherein the DNA remover is DNase type I at 5 U / ml;

[0372] · Incubate the tissue with an antimicrobial agent, wherein the antimicrobial agent is 0.1% PAA, to obtain a decellularized tissue scaffold;

[0373] · Optionally package the scaffold; and

[0374] · Subject the scaffold to ionizing radiation at about 18 kGy to 25 kGy, suitably 25 kGy to 27.5 kGy.

[0375] Those skilled in the art will recognize that the tissue can be rinsed or washed at least once between each incubation step of the method of the present invention. Suitably, the tissue can be rinsed or washed at least two or three times between each incubation step. By way of example only, the rinsing or washing can be carried out with saline. Alternatively, the rinsing or washing can be carried out with a buffer, such as a wash buffer or a well-known buffer such as PBS. Suitably, the saline can comprise a 0.9% sodium chloride solution. Suitably, the wash buffer can comprise 0.1% EDTA and 0.1% phosphate buffered saline solution. Suitably, PBS can comprise 0.1% phosphate buffered saline solution.

[0376] Specific embodiments of the present invention

[0377] In one embodiment, the method is applicable to a porcine tendon tissue scaffold. Suitably, the starting tissue is porcine tendon.

[0378] In one embodiment, the method of the present invention comprises:

[0379] (a) incubating the starting tissue with a degreasing agent;

[0380] (b) incubating the tissue with a hypotonic solution;

[0381] (c) subjecting the tissue to at least one freeze / thaw cycle;

[0382] (d) incubating the tissue with an antimicrobial agent;

[0383] (e) incubating the tissue with a hypotonic solution;

[0384] (f) incubating the tissue with an anionic detergent;

[0385] (g) incubating the tissue with a hypotonic solution;

[0386] (h) incubating the tissue with an anionic detergent;

[0387] (i) incubating the tissue with a DNA remover;

[0388] (j) incubating the tissue with a hypertonic solution;

[0389] (k) incubating the tissue with an antimicrobial agent.

[0390] In one embodiment, the degreasing agent is acetone, suitably 99% (v / v) acetone. In one embodiment, step (a) comprises incubating the starting tissue with the degreasing agent for about 2 hours. In one embodiment, step (a) comprises incubating the starting tissue with the degreasing agent in three rounds, suitably each round comprising about 40 minutes.

[0391] In one embodiment, the hypotonic solution is a hypotonic tris buffer containing 0.1% tris and 0.1% EDTA. In one embodiment, step (b) comprises incubating the tissue with the hypotonic solution for about 1 - 4 hours. In one embodiment, step (e) comprises incubating the tissue with the hypotonic solution for about 24 hours. In one embodiment, step (g) comprises incubating the tissue with the hypotonic solution for about 24 hours.

[0392] In one embodiment, the method comprises two freeze / thaw cycles. Suitably, each cycle is carried out in a hypotonic solution.

[0393] In one embodiment, the method may include a step of storing the tissue. Suitably, the tissue may be stored after being frozen. Suitably, this may be done at the end of the method, or in the middle of the method, suitably in the middle of step (c). Suitably, the storage step may last for a period of up to 12 months.

[0394] In one embodiment, the antimicrobial agent is PAA, suitably 0.1% PAA. In one embodiment, step (d) includes incubating the tissue with the antimicrobial agent for about 1 hour. In one embodiment, step (k) includes incubating the tissue with the antimicrobial agent for about 3 hours.

[0395] In one embodiment, the anionic detergent is SDS, suitably 0.1% (w / v) SDS. In one embodiment, SDS is present in a hypotonic solution, suitably present in Tris buffer. In one embodiment, step (f) includes incubating the tissue with the anionic detergent for about 24 hours. In one embodiment, step (h) includes incubating the tissue with the anionic detergent for about 24 hours.

[0396] In one embodiment, the DNA removing agent is 5 U / ml Benzonase® in a buffer, and the buffer may contain 0.6% Tris and 0.2% magnesium chloride hexahydrate. In one embodiment, step (i) includes incubating the tissue with the DNA removing agent for about 6 hours. In one embodiment, step (i) includes incubating the tissue with the DNA removing agent for three rounds, suitably each round including about 2 hours.

[0397] In one embodiment, the hypertonic solution is 0.6% Tris and 8.2% sodium chloride. In one embodiment, step (j) includes incubating the tissue with the hypertonic solution for about 18 hours.

[0398] In one embodiment, there is one or more washing steps in the method as described above. In one embodiment, the washing step may be present between step (a) and (b), between step (h) and (i), between step (i) and (j), between step (j) and (k), and / or after step (k). In one embodiment, the washing steps between step (a) and (b), between step (h) and (i), and after step (k) are carried out with saline. In one embodiment, the washing step between step (i) and (j) is carried out using a washing buffer. In one embodiment, the washing step between step (j) and (k) is carried out using PBS.

[0399] Suitably, most steps of the method are carried out at about 37°C. However, suitably, step (a) and step (b) are carried out at 18 - 30°C.

[0400] Suitably, the method further includes an end sterilization step as described elsewhere herein.

[0401] In another embodiment, the method is applicable to a porcine dermal tissue scaffold. Suitably, the starting tissue is porcine dermis.

[0402] In one embodiment, the method of the present invention includes:

[0403] (a) incubating the starting tissue with an antimicrobial agent;

[0404] (b) incubating the tissue with a degreasing agent;

[0405] (c) incubating the tissue with a protease;

[0406] (d) incubating the tissue with a degreasing agent;

[0407] (e) incubating the tissue with an anionic detergent;

[0408] (f) incubating the tissue with a hypertonic solution;

[0409] (g) incubating the tissue with a hypotonic solution;

[0410] (h) incubating the tissue with a DNA remover;

[0411] (i) incubating the tissue with an antimicrobial agent.

[0412] In one embodiment, the antimicrobial agent is PAA, suitably 0.1% PAA. In one embodiment, step (a) includes incubating the tissue with the antimicrobial agent for about 1 hour. In one embodiment, step (i) includes incubating the tissue with the antimicrobial agent for about 3 hours.

[0413] In one embodiment, the degreasing agent is acetone, suitably 99% (v / v) acetone. In one embodiment, step (b) includes incubating the starting tissue with the degreasing agent for about 33 hours. In one embodiment, step (b) includes incubating the starting tissue with the degreasing agent in three rounds, suitably the first and second rounds are about 1 hour each, and the third round is about 31 hours. In one embodiment, step (d) includes incubating the starting tissue with the degreasing agent for about 3 hours. In one embodiment, step (d) includes incubating the starting tissue with the degreasing agent in three rounds, suitably each round includes about 1 hour.

[0414] In one embodiment, the protease is a natural protease. In one embodiment, the protease is trypsin, suitably 0.2% porcine-derived trypsin in a buffer, and the buffer may contain 0.6% Tris and 0.2% magnesium chloride hexahydrate. In one embodiment, step (c) includes incubating the tissue with the protease for about 72 hours.

[0415] In one embodiment, the anionic detergent is SDS, suitably 0.01% (w / v) SDS. In one embodiment, SDS is present in the hypotonic solution, suitably present in Tris buffer. In one embodiment, step (e) comprises incubating the tissue with the anionic detergent for about 21 hours.

[0416] In one embodiment, the hypertonic solution is 0.6% Tris and 8.2% sodium chloride. In one embodiment, step (f) comprises incubating the tissue with the hypertonic solution for about 21 hours.

[0417] In one embodiment, the hypotonic solution is 0.1% Tris and 0.1% EDTA. In one embodiment, step (g) comprises incubating the tissue with the hypotonic solution for about 21 hours.

[0418] In one embodiment, the method may further comprise a step of depilating the tissue. Suitably, this step may be carried out between steps (c) and (d). Suitably, depilation may take 8 hours. Suitably, there is at least one washing step before and after the depilation step.

[0419] In one embodiment, the method may further comprise a holding step, suitably in which the tissue is held in saline. Suitably, the holding step may be carried out before the final step (i). Suitably, the holding step may be up to 72 hours.

[0420] In one embodiment, the DNA remover is Benzonase® at 5 U / ml in buffer, and the buffer may contain 0.6% Tris and 0.2% magnesium chloride hexahydrate. In one embodiment, step (h) comprises incubating the tissue with the DNA remover for about 3 hours.

[0421] In one embodiment, there is one or more washing steps in the method as described above. In one embodiment, the washing step may be present between steps (a) and (b), between steps (b) and (c), between steps (c) and (d), between steps (d) and (e), between steps (g) and (h), between steps (h) and (i), and / or after step (i). In one embodiment, the washing step is carried out with saline.

[0422] Suitably, most steps of the method are carried out at about 14°C. However, suitably, steps (a) and (i) are carried out at about 27°C. However, suitably, steps (b) and (d) are carried out at about 21.5°C.

[0423] Suitably, the method further comprises an end sterilization step as described elsewhere herein.

[0424] Decellularized tissue scaffold

[0425] The present invention provides a decellularized tissue scaffold produced by the methods described herein.

[0426] The inventors unexpectedly found that the method of the present invention removes anionic detergent (e.g., SDS) residues from the tissue. Without wishing to be bound by this hypothesis, the inventors believe that due to the optimization of the process conditions used in other steps of the method, particularly those used in incubation with hypertonic and hypotonic solutions and / or freeze / thaw cycles, the removal of anionic detergent residues is possible.

[0427] Accordingly, the decellularized tissue scaffold of the present invention is substantially free of anionic detergent (such as SDS) residues. "Substantially free" means that the tissue scaffold contains at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or up to 100% free of processing residues from anionic detergents. Suitably, by the methods described herein, the tissue scaffolds of the present invention contain fewer anionic detergent residues than those produced in the art.

[0428] Accordingly, the decellularized tissue scaffold of the present invention is substantially free of galactose-α-1,3-galactose (also known as α-gal) protein. "Substantially free" means that the tissue scaffold may include 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of α-gal compared to the amount of α-gal naturally present in the starting tissue from which the scaffold is produced. Suitably, by the methods described herein, the tissue scaffolds of the present invention contain fewer α-gal protein residues than those produced in the art.

[0429] Suitably, the tissue scaffold is a composite tissue scaffold. In one embodiment, the composite tissue scaffold is a meniscus tissue scaffold.

[0430] Examples

[0431] Example 1: Porcine dermis

[0432] Pretreatment: Dissect porcine dermis and cut it into 1 - 2 mm thick pieces.

[0433] Wash the tissue in 0.1% peracetic acid (PAA) for 1 hour, then wash with saline.

[0434] Wash the tissue three times with 99% (v / v) acetone for 1 hour, then wash three times with saline to remove acetone.

[0435] The tissue was then incubated in 0.2% trypsin for 72 hours to allow manual hair removal, and then washed three more times with saline and three times with acetone.

[0436] Decellularization: The tissue was washed in 0.01% SDS in hypotonic tris buffer for up to 24 hours, then washed with a hypertonic solution of 0.6% Tris / 9% sodium chloride for 24 hours, and further washed with hypotonic buffer (0.1% Tris / 0.1% EDTA) for 24 hours.

[0437] Wash with 5000 U / l endonuclease for 3 hours, and then wash three times in saline to remove DNA.

[0438] The tissue was cut into a certain size, then virus inactivated in 0.1% PAA, finally packaged, and finally sterilized at 25 kGy.

[0439] To demonstrate effective decellularization, the tissue was tested for DNA residue using the Qiagen - DNAeasy Blood and Tissue Kit and the NanoQuant system, and also stained with hematoxylin and eosin ( Figure 1 ). The average DNA content was confirmed to be 16 ng / mg. <50 ng / mg is considered the standard for decellularization.

[0440] Example 2: Porcine meniscus

[0441] Pretreatment: The porcine meniscus was dissected and cut into 7 - 8 mm thick slices.

[0442] The tissue was washed three times in 99% (v / v) acetone for 40 minutes each time, and then washed three times in saline to remove acetone.

[0443] The tissue was then washed in hypotonic tris buffer for 40 minutes and subjected to three freeze / thaw cycles. The tissue was frozen at -80°C.

[0444] Decellularization: The tissue was thawed and washed in 0.1% peracetic acid (PAA) for 1 hour.

[0445] The tissue was then washed in a hypertonic solution of 0.6% Tris / 9% sodium chloride for up to 8 hours, then incubated in 0.01% SDS in hypotonic tris buffer for up to 20 hours 4 times, and then washed in a hypertonic solution of 0.6% Tris / 9% sodium chloride for up to 8 hours. The tissue was then washed three times in saline for 40 minutes. Wash with 5000 U / l endonuclease twice for 2 hours, then incubate with endonuclease for 18 hours, and then wash three times with saline to remove DNA.

[0446] Processing residues were removed from the tissue by incubating the tissue in 0.01% EDTA / 0.1% phosphate buffered saline solution for up to 3 hours, followed by washing 7 times in saline and 3 times in phosphate buffered saline for 40 minutes each.

[0447] The tissue was washed in 0.1% PAA to ensure virus inactivation, followed by three washes in saline, the last saline wash for 90 hours, and then finally packaged and terminally sterilized at 25 kGy.

[0448] To demonstrate effective decellularization, the DNA residue of the tissue was tested using the Qiagen - DNAeasy Blood and Tissue Kit and the NanoQuant system, and was also stained with hematoxylin and eosin. The average DNA content was confirmed to be 14 ng / mg, while <50 ng / mg was considered the standard for decellularization.

[0449] Figure 1 Tissues were shown that achieved decellularization while maintaining the tissue architecture of the material. Figure 1 A shows the decellularized meniscus scaffold. Figure 1 B shows the decellularized dermal scaffold. In both cases, the tissue architecture of the tissue was preserved. Figure 1 C is Figure 1 Another view of the scaffold in B.

[0450] Experimental data

[0451] 1. α-gal characterization of porcine dermis

[0452] α - gal was quantified using the MyBioSource α - gal ELISA Kit (MBS262885). The tissue decellularized by the method of Example 1 was first homogenized using a high - shear mixer and extracted and processed using the standard α - gal ELISA kit method. After extraction, α - gal was quantified by measuring the absorbance of the sample at 450 nm.

[0453] Table 1

[0454]

[0455] From the results in Table 1, it can be seen that the method of the present invention significantly reduces the amount of α - gal in the tissue scaffold.

[0456] 2. Comparison of the method of the present invention with the method described in EP1392372B1

[0457] The cytotoxicity of the acellularized tissues according to EP1392372B1 and the acellularized tissues according to Example 2 herein was evaluated using ISO 10993-5 (extraction method).

[0458] Table 2

[0459]

[0460] Note: The cytotoxicity is graded from 0 - 4, with 4 being more cytotoxic. Products with a grade below 2 are acceptable as medical devices, while products with grades 3 and 4 are not acceptable as medical devices.

[0461] From the data in Table 2, it can be seen that the method of the present invention carried out in Example 2 (V2) better removes the DNA content and better removes the cytotoxic SDS residue than the prior art method.

Claims

1. A method for producing a decellularized tissue scaffold, the method comprising the steps of: · incubating the starting tissue with a degreasing agent; · incubating the tissue with an anionic detergent; · incubating the tissue with a hypotonic solution at least once, incubating the tissue with a hypertonic solution at least once, and / or subjecting the tissue to at least one freeze / thaw cycle; and · incubating the tissue with a DNA removing agent to obtain a decellularized tissue scaffold.

2. The method according to claim 1, wherein the starting tissue is selected from the group consisting of: skin, meniscus, tendon, ligament, cartilage, muscle, blood vessel and organ.

3. The method according to claim 1 or claim 2, wherein the degreasing agent is a polar solvent.

4. The method according to claim 3, wherein the polar solvent is acetone.

5. The method according to any one of the preceding claims, wherein the concentration of the degreasing agent is about 80% to about 100% (v / v).

6. The method according to any one of the preceding claims, wherein the anionic detergent is sodium dodecyl sulfate (SDS) or sodium deoxycholate.

7. The method according to any one of the preceding claims, wherein the concentration of the anionic detergent is equal to or less than 0.2% (w / v).

8. The method according to any one of the preceding claims, wherein the hypotonic solution contains 0.1% tris / 0.1% EDTA.

9. The method according to any one of the preceding claims, wherein the tissue is incubated with the hypotonic solution for 30 minutes to 100 hours, preferably 10 hours to 100 hours, preferably 20 hours to 80 hours.

10. The method according to any one of the preceding claims, wherein the hypertonic solution contains 0.6% tris / 8 - 9% sodium chloride solution.

11. The method according to any one of the preceding claims, wherein the tissue is incubated with the hypertonic solution for 1 hour to 48 hours, preferably 4 hours to 30 hours, preferably 8 hours to 24 hours.

12. The method according to any one of the preceding claims, wherein the method does not include protease inhibitors of animal origin.

13. The method according to any one of the preceding claims, wherein the method further comprises one or more steps of rinsing the tissue.

14. The method according to claim 13, wherein the method includes the step of rinsing the tissue after each of the following steps: incubating with a hypotonic solution, incubating with a hypertonic solution, and subjecting the tissue to at least one freeze / thaw cycle.

15. The method according to any one of the preceding claims, wherein the endonuclease is of human or bacterial origin, preferably wherein the endonuclease is selected from the group consisting of: DNase type I, DNase type II, DNase III, RNase, or any combination of two or more thereof.

16. The method according to claim 15, wherein the concentration of the endonuclease is about 0.5 to about 50 U / ml, preferably 5 U / ml.

17. The method according to any one of the preceding claims, Among them, the method further includes the step of incubating the tissue with an antimicrobial agent.

18. The method according to claim 17, wherein, the antimicrobial agent is an oxidizing agent, preferably wherein the oxidizing agent is peracetic acid.

19. The method according to any one of claims 17 to 18, wherein, the concentration of the antimicrobial agent is about 0.05% (w / v) to 1% (w / v).

20. The method according to any one of the preceding claims, further includes the step of subjecting the tissue to ionizing radiation, preferably wherein the dose of the ionizing radiation is about 15 to about 50 kGy, preferably about 25 kGy.

21. An acellular tissue scaffold produced by the method according to any one of claims 1 to 20.

22. The acellular tissue scaffold according to claim 21, wherein, the acellular tissue scaffold is substantially free of anionic detergent residues.

23. The acellular tissue scaffold according to claim 22, wherein, the anionic detergent residue is SDS residue.

24. The acellular tissue scaffold according to any one of claims 21 - 23, wherein, the tissue scaffold is a meniscus tissue scaffold, a dermal tissue scaffold, or a tendon tissue scaffold.

25. The acellular tissue scaffold according to claim 24, wherein, the meniscus tissue scaffold is a porcine meniscus tissue scaffold, a porcine dermal tissue scaffold, or a porcine tendon tissue scaffold.

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