Method for isolating cells from a tissue sample

Abstract

The present invention provides a method for enzymatic digestion of extracellular matrix proteins in a tissue sample, particularly comprising the steps of: preparing an aqueous medium containing (i) a matrix metalloproteinase and (ii) a cation which is Ca2+ at a concentration of 2 mmol / L or higher; and contacting the aqueous medium with a tissue sample containing an extracellular matrix protein under conditions enabling enzymatic digestion of the extracellular matrix protein, wherein the tissue sample is a cartilage tissue sample.

Description

Technical Field

[0001] The present invention relates to a method for the enzymatic digestion of extracellular matrix (ECM) proteins. The in vitro or ex vivo methods contemplated herein enable the tissue dissociation and release of cells from said tissue, which cells can then be isolated and formulated for use in medical treatment methods. The in vitro or ex vivo enzymatic digestion method of the present invention provides an improvement in the yield of isolated cells per tissue mass unit. The present invention also relates to an aqueous medium comprising a matrix metalloproteinase that can be used in applications where the disassembly of the extracellular matrix is beneficial, such as diseases associated with the pathological accumulation of the extracellular matrix, preferably diseases associated with the pathological accumulation of the extracellular matrix in the skin, on the skin, or in body openings and the associated cavities therewith. Other applications that benefit from the degradation of the extracellular matrix using an aqueous medium as disclosed herein are, for example, meat tenderization and fish boning.

Background Art

[0002] Over the past few decades, the use of cell-based medicine has been increasing. Cell-based medicine covers various fields such as cell transplantation, regenerative medicine, and tissue engineering. Examples of cell-based medicine are, for example, heart cell patches for repairing heart lesions, kidney organoids for repairing kidney function, pancreas organoids for repairing pancreas function, and intralesional injection of stromal cells from fat or bone marrow to initiate or enable a repair response. The common point of these cell-based treatment strategies is that they require the presence of a sufficient amount of tissue-isolated cells. In cell-based medical treatment methods, autologous cells are often used, whereby the cells are taken from the patient's own tissue to avoid immunological rejection. Cell isolation can be carried out in various ways, but it may include destroying the extracellular matrix of the collected tissue sample by enzymatic digestion with a matrix metalloproteinase such as collagenase to dissociate the tissue and release the cells contained in the extracellular matrix.

[0003] An example of a tissue of interest in cell-based medicine is cartilage tissue. Cartilage tissue contains chondrocytes embedded in an extracellular matrix. In healthy cartilage tissue, chondrocytes produce and maintain components of the extracellular matrix such as collagen fibrils, proteoglycans, glycosaminoglycans, and elastin. Cartilage tissue can be damaged, for example, by chondrogenesis disorders, arthritis, articular cartilage trauma, and meniscus injuries. Cartilage tissue can be repaired by autologous chondrocyte transplantation into cartilage tissue lesions. For autologous transplantation of chondrocytes, the chondrocytes first need to be isolated from a target cartilage tissue sample, for example, by degrading or destroying the extracellular matrix in the tissue sample by enzymatic digestion to release the chondrocytes. Alternatively, smaller cartilage lesions may be treated with a solution of ECM-degrading enzymes, which can result in an increase in the permeability of the ECM and subsequently an increase in the migratory potential of the resident chondrocytes around the lesion.

[0004] Matrix metalloproteinases, also known as matrix metalloproteinases, are a class of endoproteases that degrade extracellular matrix proteins by hydrolysis of peptide bonds, including several types of collagen, gelatin, elastin, fibronectin, and laminin. A particularly well-known matrix metalloproteinase is collagenase. Collagenase is known to be produced by animals, prokaryotes, fungi, and plants (Kim et al, Biochim Biophys Acta. 2007;1770(12):1627-35) and can degrade several types of collagen and gelatin.

[0005] Philominatan et al, FEBS J.2009;276(13):3589-3601 describes the effect of Ca 2+ on the conformational change of Clostridium collagenase, but the Ca 2+ concentration was substantially lower than 2 mmol / L.

[0006] Ohbayashi et al., Appl Environ Microbiol. 2012;78(16):5839-5844 reported that Ca 2+ affects the structural stability and thermal stability of Clostridial ColH, in which Ca is class II collagenase. However, it has been confirmed that Ca 2+ does not affect the digestion effect of extracellular fluid.

[0007] WO 2008 / 026928 A1 describes a cartilage repair graft. Regarding chondrocyte isolation, before subjecting a tissue sample to a digestive enzyme, the tissue sample is subjected to a treatment for increasing extracellular matrix permeability, such as contacting it with a cation including acid, base, dimethyl sulfoxide (DMSO), cathepsin, glycerol, or Na + , K + , NH 4 + , Pb 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Cd 2+ and Cu 2+ . These cations can be introduced, for example, in the form of chloride salts at a concentration of 10 mM to 2 M. After the permeability is increased, the tissue sample has been shown to be washed before being subjected to the digestive enzyme.

[0008] Collagenase is known to contain zinc and require calcium to be active (Khokha and Denhardt, Invasion Metastasis. 1989;9(6):391-405), but the effect of supra-physiological concentrations of Ca 2+ (i.e., a concentration of 2 mmol / L or higher) in an aqueous digestion medium on the effectiveness of collagenase-based ECM digestion has not been studied until now.

[0009] There is a need in the art for further methods of cell isolation, particularly methods that enable the high-yield isolation of cells from tissue samples containing extracellular matrix (ECM). There is also a need for aqueous media containing digestive enzymes that can be used in therapeutic and non-therapeutic methods where breakdown or degradation of the ECM is advantageous. SUMMARY OF THE INVENTION

[0010] Unexpectedly, the inventors have demonstrated that calcium ions at supra-physiological concentrations of 2 mmol / L or greater enable an improvement in the isolated cell yield per tissue sample when matrix metalloproteinases are incubated with tissue samples in an aqueous medium to break down the ECM and release the cells from the tissue sample.

[0011] More specifically, the inventors have surprisingly identified that simultaneous exposure of cartilage tissue samples to both matrix metalloproteinases and calcium ions at supra-physiological concentrations of 2 mmol / L or greater in an aqueous medium enables an improvement in the isolated cell yield per cartilage tissue sample.

[0012] Accordingly, the present invention provides a method for the enzymatic digestion of extracellular matrix proteins in a tissue sample, the method comprising: (i) preparing an aqueous medium comprising a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or greater; and contacting the aqueous medium with a tissue sample containing extracellular matrix proteins under conditions that enable enzymatic digestion of the extracellular matrix proteins, wherein the tissue sample is a cartilage tissue sample.

[0013] In a preferred embodiment of the method for enzymatic digestion, the aqueous medium is prepared by dissolving a calcium salt in the aqueous medium, wherein the Ca 2+a step in which the final concentration is 2 mmol / L or more, and a step of mixing matrix metalloproteinase with the aqueous medium before or after the dissolution step.

[0014] In another preferred embodiment of the method for enzymatic digestion, the step of contacting the aqueous medium with the tissue sample provides at least partial dissociation of the tissue sample, thereby releasing cells from the tissue sample in the aqueous medium.

[0015] In another preferred embodiment of the method for enzymatic digestion, the method is a method for isolating cells with or without a pericellular matrix from a tissue sample.

[0016] In another preferred embodiment of the method for enzymatic digestion, the method further comprises a step of isolating cells from the aqueous medium after enzymatic digestion, preferably by passing the digest through a cell strainer.

[0017] In another preferred embodiment of the method for enzymatic digestion, the matrix metalloproteinase is selected from the group consisting of collagenase, gelatinase, stromelysin, matrilysin, metalloelastase, enamelysin, endometase, and epilysin.

[0018] In another preferred embodiment of the method for enzymatic digestion, the matrix metalloproteinase is collagenase.

[0019] In another preferred embodiment of the method for enzymatic digestion, the matrix metalloproteinase is limited to only one matrix metalloproteinase selected from the group consisting of collagenase, gelatinase, stromelysin, matrilysin, metalloelastase, enamelysin, endometase, and epilysin.

[0020] In another preferred embodiment of the method for enzymatic digestion, the matrix metalloproteinase is limited to only collagenase.

[0021] In another preferred embodiment of the method for enzymatic digestion, collagenase is the only protease present in the aqueous medium.

[0022] In another preferred embodiment of the method for enzymatic digestion, the aqueous medium contains cations which are Ca 2+ at supra-physiological concentrations above 10 mmol / L, for example above 15 mmol / L.

[0023] In another preferred embodiment of the method for enzymatic digestion, the water-soluble medium contains dissolved CaCl 2 and.

[0024] In another preferred embodiment of the method for enzymatic digestion, the method does not include a separate incubation step of the tissue sample in a solution for increasing the permeability of the extracellular matrix, such as a cation solution, and / or a separate washing step of the tissue sample before incubating the tissue sample in the aqueous medium of the present invention. It has been established that such separate incubation steps of the tissue sample in a solution for increasing the permeability of the extracellular matrix, such as a cation solution, and / or separate washing steps before incubating the tissue sample in the aqueous medium of the present invention are not required to achieve a beneficial cell yield.

[0025] In another preferred embodiment of the method for enzymatic digestion, the tissue sample is not pancreatic tissue and / or muscle tissue, and preferably the sample is not pancreatic tissue containing pancreatic islet cells and / or muscle tissue containing smooth muscle cells.

[0026] In another preferred embodiment of the method for enzymatic digestion, the tissue sample is a tissue sample that has been collected or biopsied, and optionally, a tissue sample that has been collected or biopsied and minced prior to the step of contacting the aqueous medium with the tissue sample.

[0027] In another preferred embodiment of the method for enzymatic digestion, the cells are chondrocytes with or without their pericellular matrix, and the tissue sample is a cartilage tissue sample, preferably an articular cartilage tissue sample. Preferably, the cartilage tissue sample such as the articular cartilage tissue sample is an animal tissue sample, for example, a human tissue sample, a horse tissue sample or a bovine tissue sample.

[0028] In another aspect, the present invention provides an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation which is Ca2+ at a concentration of 2 mmol / L or higher.

[0029] In a preferred embodiment of the aqueous medium, the matrix metalloproteinase is collagenase.

[0030] In another preferred embodiment of the aqueous medium, the matrix metalloproteinase is limited to only one matrix metalloproteinase selected from the group consisting of collagenase, gelatinase, stromelysin, matrilysin, metalloelastase, enamelysin, endomethase and epilysin.

[0031] In a preferred embodiment of the aqueous medium, the matrix metalloproteinase is limited to collagenase only.

[0032] In another preferred embodiment of the aqueous medium, collagenase is the only protease present in the aqueous medium.

[0033] In another preferred embodiment of the aqueous medium, the aqueous medium contains a cation which is Ca 2+ at a supra-physiological concentration exceeding 10 mmol / L, for example exceeding 15 mmol / L.

[0034] In another preferred embodiment of the aqueous medium, the aqueous medium does not contain pancreatic tissue and / or muscle tissue, preferably the aqueous medium does not contain pancreatic tissue containing pancreatic islet cells and / or muscle tissue containing smooth muscle cells.

[0035] In another preferred embodiment of the aqueous medium, the aqueous medium further contains a cartilage tissue sample.

[0036] In another preferred embodiment of the aqueous medium, the aqueous medium further contains an articular cartilage tissue sample, chondrocytes and / or chondrons.

[0037] In another aspect, the present invention provides a container containing the aqueous medium of the present invention.

[0038] In another aspect, the present invention provides a kit containing the container of the present invention.

[0039] In another aspect, the present invention provides the use of the aqueous medium of the present invention in extracellular matrix degradation, wherein the extracellular matrix contains extracellular matrix proteins, preferably collagen.

[0040] In another aspect, the present invention provides the use of the aqueous medium of the present invention in the enzymatic digestion of extracellular matrix proteins, preferably collagen.

[0041] In another aspect, the present invention prepares the aqueous medium of the present invention for use as a medicament.

[0042] In another preferred embodiment of the aqueous medium for use as a medicament, the aqueous medium is for use in a method for treating cartilage tissue damage.

[0043] In another preferred embodiment of the aqueous medium for use as a medicament, the aqueous medium is for use in a method for treating disorders associated with the pathological accumulation of the extracellular matrix, preferably the pathological accumulation of the extracellular matrix in or on the skin or at a body orifice or in its associated cavity.

[0044] In another preferred embodiment of the aqueous medium for use as a medicament, the aqueous medium is for use in a method for treating wounds, burns, keloid disease or placental remnants, or for use in a wound debrider.

[0045] Similarly, the present invention provides a method for treating disorders associated with the pathological accumulation of the extracellular matrix, preferably the pathological accumulation of the extracellular matrix in or on the skin or at a body orifice or in its associated cavity, the method comprising administering to a subject in need thereof a therapeutically effective amount of the aqueous medium of the present invention.

[0046] In a preferred embodiment of the method for treatment, the method is a method for treating wounds, burns, keloid disease or placental remnants, or for use in a wound debrider.

[0047] Similarly, the present invention provides the use of the aqueous medium of the present invention in the manufacture of a medicament for treating disorders associated with the pathological accumulation of the extracellular matrix, preferably the pathological accumulation of the extracellular matrix in or on the skin or at a body orifice or in its associated cavity.

[0048] In a preferred embodiment of the use of the aqueous medium, the method is a method for treating wounds, burns, keloid disease or placental remnants, or for use in a wound debrider.

[0049] In another aspect, the present invention provides a method for softening meat products, comprising the steps of: preparing an aqueous medium comprising (i) matrix metalloproteinase and (ii) a cation which is Ca2+ at a concentration of 2 mmol / L or higher; and contacting the aqueous medium with a meat product comprising an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein.

[0050] In another aspect, the present invention provides a method for deboning fish products, comprising the steps of: preparing an aqueous medium comprising (i) matrix metalloproteinase and (ii) a cation which is Ca2+ at a concentration of 2 mmol / L or higher; and contacting the aqueous medium with a fish product comprising an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein.

[0051] The present invention also provides a method for enzymatic digestion of extracellular matrix proteins in a tissue sample, comprising the steps of: preparing an aqueous medium comprising (i) matrix metalloproteinase and (ii) a cation, preferably a cation of an atom which is monovalent or divalent, wherein the cation is at a supra-physiological concentration; and contacting the aqueous medium with a tissue sample comprising an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein.

[0052] Without being bound by theory, the activity of matrix metalloproteinases such as collagenase can be enhanced in swollen tissue samples. Swelling of tissue samples having many negatively charged components, such as cartilage tissue having negatively charged macromolecules, can be promoted by applying supra-physiological concentrations of calcium ions simultaneously with matrix metalloproteinases, which results in an increase in osmotic pressure and an influx of water into the tissue sample.

[0053] The present invention also provides a method for treating a subject having a cartilage tissue injury such as a meniscus injury (e.g., meniscus tear), the method comprising: obtaining or biopsying a cartilage tissue sample such as a meniscus tissue sample from a subject having a cartilage tissue injury such as a meniscus injury; optionally, mincing the tissue sample; performing the enzyme digestion disclosed herein; isolating chondrocytes and / or chondrons from the aqueous medium after the enzyme digestion; and re-seeding or transplanting the isolated (autologous) chondrocytes and / or chondrons onto or into the cartilage tissue of the subject that has been damaged, such as into or on the cartilage tissue lesion of the damaged cartilage tissue.

[0054] Similarly, the present invention provides an aqueous medium of the present invention, or a population of chondrocytes and / or chondrons isolated from said aqueous medium, for use in a method of treating a subject having a cartilage tissue injury, said method comprising: (i) an aqueous medium disclosed herein containing chondrocytes and / or chondrons (preferably, said aqueous medium is the digest disclosed herein that has passed through a strainer) or (ii) a population of isolated (autologous) chondrocytes and / or chondrons, applying, re-seeding, or transplanting onto or into the cartilage tissue of the damaged subject, such as onto or into the cartilage tissue injury (site) of the damaged cartilage tissue, preferably, said population of isolated chondrocytes and / or chondrons is obtained by a method comprising: providing (or obtaining or biopsying) a cartilage tissue sample such as a meniscus tissue sample from a subject having a cartilage tissue injury such as a meniscus injury (e.g., torn meniscus); performing the method for enzyme digestion of the present invention; and isolating chondrocytes and / or chondrons from the aqueous medium after the enzyme digestion.

[0055] In another aspect, the present invention provides a method for generating a peptide or protein hydrolysate, the method comprising: preparing an aqueous medium of the present invention; and contacting the aqueous medium with a composition containing a protein under conditions that allow for enzymatic digestion of the protein.

[0056] In a preferred embodiment of the method, the peptide is a bioactive peptide such as a collagen peptide, and the protein is collagen.

[0057] In another preferred embodiment of the method, the protein hydrolyzate is a collagen hydrolyzate, and the protein is collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0058]

Figure 1

Figure 2

[0059] Definitions As used herein, the term “a” or “an,” for example with respect to matrix metalloproteinase, includes reference to one or more matrix metalloproteinases.

[0060] As used herein, the term "enzymatic digestion" includes reference to the chemical conversion or subdivision of macromolecules by enzymatic activity. Preferably, this term relates to the proteolytic cleavage of peptide bonds in proteins by enzymes such as matrix metalloproteinases. Preferably, the enzymatic digestion disclosed herein includes the hydrolysis of proteins.

[0061] As used herein, the term "extracellular matrix" includes reference to a network of macromolecules and minerals that provides structural support and / or protection to cells in its direct or indirect vicinity and interacts with cells via cell surface receptors. The extracellular matrix includes the interstitial matrix and the basement membrane. The major components of the animal extracellular matrix include proteoglycans, non-proteoglycan polysaccharides, extracellular vesicles, and extracellular matrix proteins. The extracellular matrix can embed various cell types depending on the tissue type.

[0062] As used herein, the term "extracellular matrix protein" includes reference to proteins that are part of the extracellular matrix. In animals, extracellular matrix proteins include, among others, collagen, elastin, fibronectin, laminin, actin, and integrin. Preferably, the extracellular matrix disclosed herein is the extracellular matrix of an animal such as a human tissue sample. Preferably, the extracellular matrix protein disclosed herein is collagen.

[0063] As used herein, the term "tissue" includes reference to the cellular tissue level between cells and complete organs. This term includes reference to cells having their extracellular matrix that perform a particular function together. Non-limiting examples of tissues include connective tissue, epithelial tissue, muscle tissue, and nerve tissue.

[0064] As used herein, the term "tissue sample" includes reference to tissue obtained from a subject, for example, by collection or biopsy. A tissue sample can be obtained from an animal subject, such as a human subject. A tissue sample is generally a tissue portion that includes cells having their extracellular matrix. One of ordinary skill in the art will understand that the methods for enzymatic digestion disclosed herein can be performed in vitro or ex vivo, for example, when it is necessary to isolate cells for subsequent autologous or allogeneic transplantation. Alternatively, the aqueous media disclosed herein can be used for the treatment of diseases associated with the pathological accumulation of extracellular matrix. This term also includes reference to any other non-animal tissue containing extracellular matrix.

[0065] As used herein, the terms "incubate" and "incubation" include reference to the active or passive facilitation of contact between two or more chemical substances, mixtures, solutions, suspensions, emulsions, extractants, crystals, sols, colloids, gels, biological materials, tissues, media, or combinations thereof. No limitation as to the length of the period is intended. External conditions such as temperature, pH, and agitation can be part of the incubation process. Incubation enables reactions, such as enzymatic digestion reactions, to occur. Incubation can be performed with or without using an incubator vessel. As used herein in connection with incubating a tissue or tissue sample with the aqueous media disclosed herein, the term "incubation" can be used interchangeably with the phrase "contacting the tissue sample with the aqueous media under conditions that enable enzymatic digestion of the extracellular matrix proteins." One of ordinary skill in the art is familiar with the appropriate conditions that enable enzymatic digestion.

[0066] The term "aqueous medium", as used herein, includes reference to a liquid aqueous medium, e.g., a liquid medium that contains water as a main component. The medium may further contain salts, anions, cations, carbohydrates, polymers, cells, fats, fatty acids, other solvents, any other organic or inorganic components, or combinations thereof. Preferably, the aqueous medium is a liquid aqueous medium such as a solution, suspension or colloid.

[0067] The term "cation", as used herein, includes reference to a positively charged ion. A cation can be, for example, a monovalent or divalent atomic ion, e.g., a monovalent or divalent metal ion. Preferably, the cation is a divalent metal ion such as Ca 2+ Other cations that can be applied in the present invention are Na + , K + , NH 4 + , Pb 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Fe 2+ , and Cu 2+ or Co 2+ , Se 4+ , or Cr 3+ .

[0068] The term "concentration", as used herein, includes reference to the molar concentration (mol / L) of a compound in an aqueous medium. In the context of a cation in an aqueous medium, the concentration refers to the concentration of the cation prior to the steps disclosed herein of contacting the aqueous medium with a tissue sample, meat product or fish product.

[0069] The term "matrix metalloproteinase (MMP)", as used herein, includes reference to a group of endoproteases that hydrolyze extracellular matrix proteins and whose function is dependent on the presence of at least one metal such as calcium. Matrix metalloproteinases (MMPs) are also referred to as matrix metallopeptidases or matrilysins. Preferably, an MMP is a collagenase, gelatinase, stromelysin, matrilysin, metalloelastase, enamelysin, endometase or epilysin. The group of matrix metalloproteinases includes the gene products of the human genes MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28, their post-translational modifications and / or active forms, and all their homologues, orthologues, paralogues and counterparts in animals, plants, fungi, other eukaryotes, bacteria, archaea and viruses. More preferably, the matrix metalloproteinase is a collagenase such as MMP1, MMP2, MMP8, MMP9 or MMP13.

[0070] The term "mixing", as used herein in connection with the mixing of an MMP with an aqueous medium, includes mixing an MMP (which is solid, e.g., provided in the form of a lyophilized, non-sterile powder or pre-dissolved in a liquid medium) with the aqueous medium by, for example, adding the MMP in solid or dissolved form to the aqueous medium while gently stirring.

[0071] As used herein in the context of dissociation of tissue samples during enzymatic digestion, the term "dissociation" includes reference to at least partial loss of integrity of the tissue sample caused by enzymatic digestion of ECM proteins in the tissue sample and release of cells from the tissue sample. Cells released from the tissue sample tend to no longer be contained within the tissue sample, but may still be attached to other components that were previously part of the tissue, such as other cells and / or the pericellular matrix. An example of dissociation of a tissue sample is at least partial dissociation of a cartilage tissue sample upon enzymatic digestion and release of chondrocytes from the cartilage tissue sample, with or without their pericellular matrix. As used herein, the term "cell" includes reference to cells with or without a pericellular matrix. Preferred cells are chondrocytes or chondrons. A chondron is a chondrocyte with a pericellular matrix. When reference is made herein to chondrocytes, it includes reference to chondrons.

[0072] As used herein, the term "collagenase" includes reference to enzymes belonging to the group of matrix metalloproteinases (MMPs). Collagenase is known to cleave peptide bonds in collagen and procollagen, among other things. Preferably, collagenase cleaves peptide bonds by hydrolysis. The genus of collagenase includes, inter alia, interstitial collagenase, neutrophil collagenase, collagenase 3, collagenase 4, xenopus collagenase, fibroblast-type collagenase, PMN-type collagenase, type IV collagenase, ColH, ColG, AUX-I and AUX-II. The collagenase disclosed herein may be of animal, plant, fungal, bacterial or archaeal origin and may be used in purified, partially purified or unpurified form in the methods of the present invention. Collagenase for use in the methods disclosed herein may be produced commercially or non-commercially. Potential substrates of collagenase include any type of collagen, including type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type IX collagen, type X collagen, type XI collagen, type XIIA collagen, type XIIB collagen, type XIV collagen, type XVI collagen, type XXII collagen and type XXVI collagen. Another non-limiting example of a potential substrate of collagenase is gelatin. Preferably, the collagenase is a microbial collagenase, preferably a bacterial collagenase.

[0073] As used herein, the term "gelatinase" includes reference to enzymes belonging to the group of matrix metalloproteinases (MMPs). Gelatinases can cleave peptide bonds in, but not limited to, collagen, procollagen, and gelatin. The genus of gelatinases includes, inter alia, gelatinase A and gelatinase B. Gelatinases can be of animal, plant, fungal, bacterial, or archaeal origin and can be used in purified, partially purified, or unpurified form. Gelatinases for use in the methods of the present invention can be produced commercially or non-commercially. Non-limiting examples of substrates for gelatinases include type I gelatin, type V gelatin, type I collagen, type III collagen, type IV collagen, type V collagen, type VII collagen, type X collagen, type XI collagen, tenascin, elastin, fibronectin, laminin-5, vitronectin, and entactin.

[0074] As used herein, the term "stromelysin" includes reference to enzymes belonging to the group of matrix metalloproteinases (MMPs). Stromelysins can cleave peptide bonds in, but not limited to, collagen and procollagen. The genus of stromelysins includes, inter alia, stromelysin-1, stromelysin-2, stromelysin-3, and stromelysin-4 (also called RASI-1). Stromelysins can be of animal, plant, fungal, bacterial, or archaeal origin and can be used in purified, partially purified, or unpurified form. Stromelysins for use in the methods of the present invention can be produced commercially or non-commercially. Non-limiting examples of substrates for stromelysins include type II collagen, type IV collagen, type IX collagen, type X collagen, type XI collagen, gelatin, laminin, fibronectin, elastin, and aggrecan.

[0075] As used herein, the term "matrilysin" includes reference to enzymes belonging to the group of matrix metalloproteinases (MMPs). Matrilysin can cleave peptide bonds of, but not limited to, collagen and procollagen. The matrilysin genus includes, among others, matrilysin 1 (also referred to as PUMP-1 or uterine metalloproteinase) and matrilysin 2 (also referred to as endometase). Matrilysin may be of animal, plant, fungal, bacterial or archaeal origin and may be used in purified, partially purified or unpurified form. Matrilysin for use in the methods of the present invention can be produced commercially or non-commercially. Non-limiting examples of substrates for matrilysin include type III collagen, type IV collagen, type V collagen, type IX collagen, type X collagen, type XI collagen, type I gelatin, type II gelatin, type IV gelatin, type V gelatin, fibronectin, proteoglycan, fibrinogen, casein and vitronectin.

[0076] As used herein, the terms "metalloelastase" and the expression "macrophage metalloelastase" include reference to enzymes within the group of matrix metalloproteinases. Metalloelastase can cleave peptide bonds in, but not limited to, elastin. Metalloelastase may be of animal, plant, fungal, bacterial or archaeal origin and may be used in purified, partially purified or unpurified form. Metalloelastase for use in the methods of the present invention can be produced commercially or non-commercially. Non-limiting examples of substrates for metalloelastase include elastin, type IV collagen, type I gelatin, fibronectin, laminin, vitronectin and proteoglycan.

[0077] As used herein, the term "enamelysin" includes reference to enzymes within the group of matrix metalloproteinases (MMPs). Enamelysin can, but is not limited to, cleaving peptide bonds in amelogenin. Enamelysin may be of animal, plant, fungal, bacterial or archaeal origin and may be used in purified, partially purified or unpurified form. Enamelysin for use in the methods of the present invention may be produced commercially or non-commercially. Non-limiting examples of substrates for enamelysin include amelogenin and ameloblastin.

[0078] As used herein, the term "epilysin" includes reference to proteins within the group of matrix metalloproteinases (MMPs). Epilysin can, but is not limited to, cleaving peptide bonds in casein, but not exclusively. Epilysin may be of animal, plant, fungal, bacterial or archaeal origin and may be used in purified, partially purified or unpurified form. Epilysin for use in the methods of the present invention may be produced commercially or non-commercially.

[0079] As used herein, the term "dissolving" or "dissolve" includes reference to the formation of a solution comprising at least one solvent and at least one solute.

[0080] The terms "cartilage tissue" or "cartilaginous tissue" can be used interchangeably and include references to tissue from cartilage. Cartilage tissue may include tissue from types of elastic cartilage, hyaline cartilage, and / or fibrocartilage. Preferably, the cartilage tissue is hyaline cartilage tissue. Cartilage tissue can be derived from any type of animal cartilage, including, but not limited to, joints, the thorax, ears, nose, tracheobronchi, and intervertebral discs. Cartilage tissue includes chondrocytes and an extracellular matrix in which the chondrocytes are embedded. The biochemical composition of cartilage tissue depends on its location (and, if applicable, its location prior to harvesting or biopsy) and type, but common features include collagen fibers, elastin fibers, aggrecan, glycosaminoglycans, and proteoglycans. Type II collagen is the most prominent type of collagen in cartilage, and other types of collagen that can be found in cartilage are type IV collagen, type VI collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIV collagen, type XIV collagen, type XXII collagen, and type XXVII collagen.

[0081] Cartilage, preferably hyaline cartilage, contains negatively charged components such as chondroitin-4-sulfate, chondroitin-6-sulfate, heparan sulfate, and keratan sulfate. The structure and composition of the extracellular matrix of cartilage are significantly different from those of other tissues such as pancreatic tissue and muscle tissue. The extracellular matrix of cartilage contains, for example, significantly more type II collagen and glycosaminoglycans (GAGs) than that of pancreatic tissue and / or muscle tissue. Furthermore, the extracellular matrix of cartilage is, for example, more dense and more tightly packed than that of pancreatic tissue and / or muscle tissue. Whereas the extracellular matrix represents the main functional component of cartilage, cells represent the main functional component in other tissues such as pancreatic tissue and / or muscle tissue.

[0082] As used herein, the term "cardiac tissue" includes references to tissue from the heart. Cardiac tissue can include ventricular cardiomyocytes, atrial cardiomyocytes, smooth muscle cells, pericytes, fibroblasts, mesothelial cells, endothelial cells, adipocytes, and / or cardiac pacemaker cells. Cardiac tissue preferably includes an extracellular matrix. Notable components of the extracellular matrix of cardiac tissue can be type I collagen and type III collagen.

[0083] As used herein, the term "liver tissue" includes references to tissue from the liver. Liver tissue can include hepatocytes, Kupffer cells, hepatic stellate cells, and hepatic sinusoidal endothelial cells. Liver tissue preferably includes an extracellular matrix. Notable components of the extracellular matrix of liver tissue generally are type I collagen, type III collagen, type IV collagen, and type V collagen.

[0084] As used herein, the term "pancreatic tissue" includes references to tissue from the pancreas. Pancreatic tissue can include alpha cells, beta cells, delta cells, epsilon cells, PP cells (also called gamma cells), and / or pancreatic connective tissue cells. Pancreatic tissue preferably includes an extracellular matrix. Notable components of the extracellular matrix of pancreatic tissue are type I collagen, type III collagen, type IV collagen, and type V collagen.

[0085] As used herein, the term "thyroid tissue" refers to tissue derived from the thyroid. Thyroid tissue can include follicular cells and / or parafollicular cells. Thyroid tissue preferably includes an extracellular matrix. Notable components of the extracellular matrix of thyroid tissue are type I collagen and type III collagen.

[0086] As used herein, the term "salivary gland tissue" includes references to tissue from one of the salivary glands. Salivary gland tissue can include serous cells, mucous cells, acinar cells, duct cells, and / or myoepithelial cells. Thyroid tissue preferably includes an extracellular matrix. Notable components of the extracellular matrix of thyroid tissue is type I collagen.

[0087] The terms "harvested" or "biopsied", as used herein, include reference to obtaining a tissue sample from a subject by methods and means generally known in the art.

[0088] The term "subject", as used herein, preferably relates to a mammal such as a human. Other subjects can be non-human primates, domestic animals such as dogs, cats, sheep, cows, goats, pigs, horses, experimental animals such as mice, rats, rabbits, guinea pigs, and captive animals such as zoo animals. In a preferred embodiment, the subject is (i) a human, such as a human patient, suffering from or suspected of suffering from a disorder associated with a pathological accumulation of extracellular matrix, preferably a pathological accumulation of extracellular matrix in or on the skin or in or associated with a body orifice or cavity thereof, e.g., a wound, burn, keloid disorder or placental remnant, or (ii) a human suffering from a cartilage tissue injury disclosed herein. A pathological accumulation of extracellular matrix means, inter alia, an excessive growth or deposition of ECM that, if left untreated, provides an adverse health condition.

[0089] The terms "minced" or "mincing", as used herein, include reference to the mechanical division of a tissue sample, such as a harvested or biopsied tissue sample, e.g., by cutting with a scalpel. One of ordinary skill in the art knows the generally known methods and means for mincing a tissue sample.

[0090] The term "isolating", as used herein in connection with isolating cells from a tissue sample, includes reference to the release of cells from a tissue sample as a result of applying the methods of the present invention for enzymatic digestion, and / or the subsequent recovery of cells from the digest, e.g., by passing the digest through a cell strainer.

[0091] As used herein, the term "digestate" includes reference to a composition obtained after contacting an aqueous medium with a tissue sample comprising an extracellular matrix protein disclosed herein, i.e., an enzymatically digested ECM protein, an at least partially dissociated tissue sample and / or an aqueous composition comprising cells released from said tissue sample.

[0092] As used herein, the term "chondrocyte" includes reference to a cell that produces components of cartilage tissue and / or maintains the cartilage extracellular matrix. This term includes chondrons, which are chondrocytes having a pericellular matrix.

[0093] As used herein, the expression "articular cartilage tissue" includes reference to a specific type of cartilage tissue found on the surface of a bone having a synovial joint. Articular cartilage is an example of hyaline cartilage. Articular cartilage tissue can be obtained from any suitable source, such as a meniscus.

[0094] As used herein, the term "treatment" includes reference to the application or administration to a subject of an agent or composition comprising an agent, for the purpose of partially or completely reversing, alleviating, and / or inhibiting the progression of a disease or its symptoms.

[0095] As used herein, the term "therapeutically effective amount" includes reference to an amount having a therapeutic effect or required to produce a therapeutic effect in a subject. For example, a therapeutically effective amount of the aqueous composition of the present invention is an amount required to produce the desired therapeutic effect, which will be determined by clinical trial results, model animal studies, and / or in vitro studies. The therapeutically effective amount can depend on several factors including, but not limited to, the characteristics of the subject (e.g., height, weight, gender, age and medical history) and the type of disease.

[0096] As used herein, the term "wound" includes reference to any type of skin tissue injury associated with a pathological accumulation of extracellular matrix.

[0097] As used herein, the term "thermal injury" includes reference to skin wounds caused by heat, chemical or radiation exposure, or abrasive friction. The injury may be superficial or may affect deeper layers of the skin and underlying tissues. Preferably, thermal injury is accompanied by a pathological accumulation of extracellular matrix.

[0098] As used herein, the term "keloid disorder" includes reference to any disorder in which excessive growth of scar tissue is involved and can extend beyond the boundaries of the original skin injury or lesion.

[0099] As used herein, the term "retained placenta" includes reference to a disorder in which the placenta remains partially or completely in the uterus after (vaginal) birth. Generally, the placenta remains when it is not expelled within 30 minutes after birth. Retained placenta can be treated, for example, by intraplacental administration of the aqueous composition of the present invention or by umbilical cord injection of the aqueous composition of the present invention.

[0100] As used herein, the term "wound debrider" includes reference to enzymatic wound debridement, which is the standard of care for non-healing and necrotic wounds in subjects where surgical intervention is not an option. Preferably, the subject is a subject having a non-healing or necrotic wound and optionally also a subject where surgical intervention is not an option. Collagenase is described, for example, as an enzymatic debriding product selected for use in subjects having wounds containing bacteria, including yellow slough and / or eschar. Preferably, the wound debrider is enzymatic wound debridement.

[0101] The term "softening", as used herein, includes a process of making meat products easier to chew or easier to cut. Generally, the disassembly or breakdown of extracellular matrix proteins such as collagen in meat products provides a softer meat product. Methods of softening meat include mechanical softening, enzymatic softening, acid softening, salt solutions, sodium bicarbonate and / or heat. Preferably, in the method of the present invention, softening is enzymatic softening.

[0102] The term "meat product", as used herein, includes reference to meat from slaughtered animals or artificial meat that is suitable for human consumption or can be made suitable for human consumption. Meat products generally include muscle tissue and / or adipose tissue. Meat products can be meat products derived from pigs, cows, sheep, chickens, turkeys, goats, ducks and / or deer.

[0103] The term "boning", as used herein, includes reference to the process of separating fish meat from fish bones. Boning of fish products can be done before or after preparation for consumption. Preferably, in the method of the present invention, the fish product is a fish with a head, eviscerated, and / or filleted, and optionally further tail-cutted.

[0104] The term "fish product", as used herein, includes reference to a product that includes both the flesh (fillet) and bones of a fish. Preferably, the fish product is a fish with a head, eviscerated, and / or filleted, and optionally further tail-cutted.

[0105] The phrase "body opening or associated cavity", as used herein, includes reference to any body opening and the extracellular cavity associated therewith. Examples of body openings and associated cavities include the nostrils and nasal cavity, the oral cavity and pharynx, the ear opening and external auditory canal, the anus, and the intestines, such as the rectum, the urethral opening, the urethra, the bladder, the vagina, and the uterus.

[0106] As used herein, the term "supraphysiological concentration" includes reference to the concentration of cations such as Ca 2+ in the aqueous medium of the present invention, where said concentration is higher than the concentration of said cation that the tissue normally experiences before being enzymatically digested. For example, for Ca 2+ , the physiological concentration in various tissue types does not exceed 2 mmol / L. For other cations, the trace element concentration ranges in mammalian serum are noted to be 0.0179 - 0.0358 mmol / L for iron, 0.0122 - 0.0184 mmol / L for zinc, 0.00897 - 0.0157 mmol / L for copper, 0.170 - 0.509 μmol / L for cobalt, 3.28 - 3.46 μmol / L for manganese, and 0.633 - 2.79 nmol / L for selenium (Yatoo et al, Veterinary World. 2013;6(12):963 - 967). Cation concentrations of these elements in the aqueous medium of the present invention that exceed these ranges (e.g., as measured in serum) would be considered supraphysiological.

[0107] As used herein, the term "bioactive peptide" includes reference to peptides that exhibit effects that are preferably beneficial to the human or animal body as a result of their bioactive properties. Bioactive peptides typically derive from foods such as protein-containing materials from plants and / or animal products, including grains, milk, eggs, soybeans, fish, and meat. Bioactive peptides are typically obtained by hydrolysis of the whole protein. Beneficial effects of bioactive peptides include, but are not limited to, affecting human or animal metabolism, preventing diseases, reducing chronic diseases, having a blood pressure-lowering effect, an antibacterial effect, an antithrombotic effect, an immunomodulatory effect, and an effect on mineral-binding function. Bioactive peptides can be produced enzymatically, via food processing, or by microbial fermentation. Preferably, the compositions containing the proteins disclosed herein contain collagen (i.e., as an enzymatically digestible protein that can be enzymatically digested by the matrix metalloproteinase disclosed herein in collagen peptides). Further, the compositions containing the proteins disclosed herein can be whole-protein preparations.

[0108] As used herein, the term "protein hydrolysate" includes reference to the whole protein after digestion of protein fragments. Protein hydrolysates can be taken up more easily and rapidly by the human or animal body compared to whole proteins. Protein hydrolysates are also known in relation to cell culture.

[0109] As used herein, the term "whole-protein preparation" includes reference to proteins that are not completely digested and preferably (still) not partially digested. These proteins can form the basis for the production of bioactive peptides and / or protein hydrolysates.

[0110] Method for isolating cells from tissue by enzymatic digestion of ECM Isolation of cells from tissues has been found to be difficult and does not always result in the desired amount of cells. This is particularly relevant for tissue types where the presence of the extracellular matrix prevents the release and isolation of cells from the tissue. Examples of such tissues are cartilage tissues, which contain large amounts of glycosaminoglycans, proteoglycans, collagen and elastin fibers. For multiple administrations of cell-based pharmaceuticals, isolation of cells from tissues is necessary. If the isolation process is inefficient, it becomes necessary to provide larger tissue samples to generate a sufficient number of isolated cells.

[0111] The present invention provides a method for efficiently isolating cells from a tissue sample by enzymatically digesting ECM proteins in the tissue sample using an aqueous medium containing a matrix metalloproteinase and a cation such as Ca 2+ This enzymatic digestion method can be used, inter alia, to isolate chondrocytes and / or chondrons from cartilage tissue. The method of the present invention relates specifically to an improved digestion of extracellular matrix proteins in the extracellular matrix of a tissue sample by a matrix metalloproteinase, which is also suitable for other processes that require the degradation of extracellular matrix proteins, such as the treatment of wounds, burns, keloid diseases or placental remnants, enzymatic wound debridement, meat softening or fish boning. The advantageous use of matrix metalloproteinases such as collagenase in such methods is already established. For example, with regard to enzymatic wound debridement, collagenase is the standard treatment for non-healing and necrotic wounds in subjects where surgical intervention is not an option.

[0112] Preferably, in the method of the present invention for enzymatic digestion or isolation of cells from tissue, the first step involves providing a tissue sample from a subject, for example, by collection or biopsy. Such a subject can be a subject having cartilage tissue damage such as a lesion of articular cartilage. Those skilled in the art are well aware of the appropriate methods and means for providing a tissue sample. Furthermore, such a step of collecting a tissue sample can be intraoperative, i.e., can be part of a method for treating a subject having cartilage tissue injury such as an articular cartilage lesion as disclosed herein.

[0113] The collected or biopsied tissue is optionally minced prior to enzymatic digestion to increase the surface area of the tissue. Mincing can be carried out, inter alia, by cutting the collected or biopsied tissue sample with any suitable instrument such as a scalpel.

[0114] Subsequently, the tissue sample is contacted with an aqueous medium containing matrix metalloproteinase and at least one cation, for example Ca 2+ at a supra-physiological concentration (i.e., a concentration higher than the normal physiological concentration of the cation in the tissue or serum) under conditions that allow enzymatic digestion of extracellular matrix proteins in the tissue sample. After an appropriate incubation time, under appropriate incubation conditions, the resulting digest can be sieved, for example, using a cell strainer with a pore size of 100 μm, to filter out undigested tissue. The sieved digest can be washed with an appropriate solution such as phosphate buffered saline to wash away or remove the matrix-metalloproteinase-containing medium. A representative sample can be taken from the washed digest (which is a suspension of individual primary cells and clustered primary cells having various amounts of remaining extracellular matrix and extracellular matrix fragments) to evaluate the cell concentration of the digest. The terms "contact" and "incubate" can be used interchangeably herein.

[0115] Ca2+ The physiological concentration can vary between organs and tissue types. For example, in mammals, the extracellular Ca 2+ physiological concentration is 1 - 2 mmol / L. The Ca 2+ in human serum is, for example, 1.1 - 1.3 mmol / L. The Ca 2+ concentration in human synovial fluid is 0.32 mmol / L. Physiological concentration means the concentration under homeostatic conditions in a living, healthy animal such as a human or in a tissue of interest within the animal, and preferably, the physiological concentration is the extracellular concentration of the cations disclosed herein under homeostatic conditions in a living, healthy animal such as a human or in a tissue of interest within the animal. Supraphysiological concentration is a concentration that exceeds the physiological concentration in or within the tissue of interest. Since the Ca 2+ concentration in a living, healthy animal is not more than 2 mmol / L, a supraphysiological Ca 2+ concentration is recognized as being at least 2 mmol / L, or higher than 2 mmol / L. In embodiments, the aqueous medium preferably contains Ca 2+ at 2 mmol / L or higher, or higher than 2 mmol / L, more preferably 2 - 2000 mmol / L, more preferably 2 - 200 mmol / L, even more preferably 2 - 50 mmol / L, most preferably 3 - 10 mmol / L, for example 3, 4, 5, 6, 7, 8, 9 or 10 mmol / L of Ca 2+ and may contain Ca 2+ at 10 mmol / L or higher. The aqueous medium may contain Ca 2+ at more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 mmol / L. The aqueous medium may contain Ca 2+ at 15 - 50, 15 - 40, 15 - 30 or 15 - 20 mmol / L. Cations such as Ca 2+ include CaSO 4 , calcium citrate, CaCO 3 , CaBr 2 , CaF 2 , CaI 2 , CaCl 2 , Ca(NO 3 )2 , CaC 2 O 4 , Ca(H 2 PO 4 ) 2 or CAHPO 4 in the form of any salts such as can be added to the aqueous medium. Most preferably, the cation which is Ca 2+ is added to the aqueous medium in the form of its CaCl 2 salt.

[0116] In the method of the present invention, the enzymatic digestion is carried out by one or more matrix metalloproteinases (MMPs) contained in the aqueous medium disclosed herein. Examples of MMPs are collagenase, gelatinase, stromelysin, matrilysin, metalloesterase, enamelysin, endometase and epilysin. The MMPs used in the method of the present invention include MMPs that are of animal, plant, fungal or bacterial origin, as well as homologs, orthologs, paralogs and counterparts thereof such as MEROPS peptidase family M9 enzymes. Examples of MEROPS peptidase family M9 enzymes are BP, ColH and ColG from Clostridium histolyticum, ColH and class II collagenase from Bacillus cereus, M9A protein and bacterial collagenase V from Vibrio alginolyticus, VMC peptidase from Vibrio mimicus, ColT and class I collagenase from Clostridium tetani, subfamily M9B protein from Microscilla marina, collagenases from Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Rhizoctonia solani, Thermoactinomyces spE-21 or Clostridium perfringens. Preferably, the MMPs disclosed herein are collagenases. Two or more types of collagenase can be used in the method of the present invention. More preferably, type II collagenase is used. The amount of MMP in the aqueous medium disclosed herein is preferably 0.2% w / v to 10% w / v. Preferably, the amount of MMP in the aqueous medium is about 2% w / v.

[0117] The aqueous media disclosed herein may further comprise a buffer to maintain a pH corresponding to a physiological pH, such as about 7. The buffer may be present at a concentration of 1 to 100 mM. Examples of suitable buffers include organic and inorganic compounds and their salts, such as citrate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, phosphate, carbonate, bicarbonate, and borate buffers. Additionally, the aqueous media may include amino acid-based buffers, urea buffers, and buffers such as Tris, MOPS, ACES, PIPES, BES, and HEPES.

[0118] Other components that may be included in the aqueous media as disclosed herein include DTPA, EDTA, EGTA; reducing agents such as dithiothreitol, dithioerythritol, β-mercaptoethanol, glutathione, thioredoxin, cysteine, ascorbic acid, thioglycolate; MgCl 2 , NaCl, KCl, ZnCl 2 , ZnSO 4 ions necessary for the activation of any of the other components in the media; organic solvents such as DMSO or lipid / membrane modifiers; non-ionic surfactants such as Triton X-100; and / or osmoprotectants such as sucrose.

[0119] In a preferred embodiment, the enzymatic digestion method of the present invention comprises the steps of preparing an aqueous media; dissolving a calcium salt in the aqueous media, wherein the final concentration of Ca 2+ in the aqueous media is 2 mmol / L or greater; and mixing a matrix metalloproteinase with the aqueous media before or after the dissolving step. Preparation can be carried out by adding the stock components together to reach the desired concentration and by mixing the components appropriately and properly. Mixing can be carried out by pipetting, shaking, stirring, vortexing or mixing, sonication, and / or passive mixing. Preferably, mixing is carried out by vortexing. Preferably, the mixing of the aqueous media is carried out for a duration between 0.1 and 20 minutes.

[0120] In the method of enzymatic digestion of the present invention, a tissue sample containing extracellular matrix protein is mixed with the aqueous medium disclosed herein. The mixing can be carried out by pipetting, shaking, stirring, vortexing or mixing, sonication and / or passive mixing. Preferably, the tissue sample and the medium are mixed by vortexing.

[0121] The incubation or contact of the aqueous medium described herein with the tissue sample means that the tissue sample is at least partially, preferably completely, immersed in the aqueous medium. During incubation, the tissue sample is in contact with the aqueous medium and thereby with its components. During incubation, the container containing the aqueous medium and the tissue sample can be shaken. Preferably, the incubation is carried out under continuous shaking. The temperature during incubation can be adjusted. Preferably, the temperature during incubation is constant. More preferably, the temperature during incubation is constant at about 37 °C. The incubation can be carried out for 1 to 120 minutes. Preferably, the incubation is carried out for 5 to 60 minutes. More preferably, the incubation takes about 35 minutes.

[0122] In certain embodiments, the method of the present invention includes isolating or separating the cells from the aqueous medium by passing the digest through a cell strainer having a pore size such that preferably cells pass through and larger components are blocked after enzymatic digestion. Preferably, the cell strainer has a pore size of about 100 μm.

[0123] The tissue samples disclosed herein can be of any size or shape. Preferably, it is a fragment or sub-component of a tissue containing ECM found in the body of an animal. The tissue sample is preferably an animal tissue sample, more preferably the tissue sample is a human tissue sample.

[0124] Preferably, the tissue samples disclosed in this specification are selected from the group consisting of samples of cartilage tissue, heart tissue, liver tissue, pancreatic tissue, thyroid tissue, salivary gland tissue, bone tissue, skin tissue, kidney tissue, lung tissue, adipose tissue, tendon tissue, bladder tissue, stomach tissue, colon tissue, esophageal tissue, muscle tissue, nerve tissue, connective tissue, and epithelial tissue. More preferably, the tissue sample is selected from the group including cartilage tissue, heart tissue, liver tissue, pancreatic tissue, thyroid tissue, and salivary gland tissue.

[0125] Even more preferably, the tissue sample is a cartilage tissue sample. There are mainly three types of cartilage tissue: elastic cartilage, hyaline cartilage, and fibrocartilage tissue. Preferably, the cartilage tissue is hyaline cartilage tissue. More preferably, the cartilage tissue is articular cartilage such as meniscus cartilage tissue.

[0126] The articular cartilage tissue disclosed in this specification can be collected or biopsied from any appropriate location within the animal body. Examples of locations within the animal body suitable for cartilage tissue collection include, for example, the middle finger interphalangeal joint, the middle toe interphalangeal joint, the interphalangeal joint, the midtarsal joint, the calcaneocuboid joint, the cuneiform joint, the cuboid navicular joint, the cuneonavicular joint, the calcaneocuboid joint, the talocalcaneonavicular joint, the subtalar joint, the talocrural joint, the tibiofibular joint, the patellofemoral joint, the tibiofemoral joint, the hip joint, the sternoclavicular joint, the acromioclavicular joint, the shoulder joint, the radiocarpal joint, the ulnar carpal joint, the proximal radioulnar joint, the distal radioulnar joint, the radiocarpal joint, the intercarpal joint, the central carpal joint, the carpometacarpal joint, the metacarpophalangeal joint, the interphalangeal joint of the finger, the atlantoaxial joint, the intervertebral joint, the lumbosacral joint, the sacrococcygeal joint, the costovertebral joint, the sternocostal joint, the interchondral joint, the costochondral joint of the rib, the symphysis pubis, the sacroiliac joint, the temporomandibular joint, and the atlantooccipital joint. Preferably, the cartilage is collected from the middle finger interphalangeal joint and / or the middle toe interphalangeal joint.

[0127] Preferably, in a method for treating a subject with cartilage tissue injury, the cartilage tissue sample is collected from the same tissue as the tissue including the cartilage tissue injury (which may also be referred to as a defect or lesion). For example, when the cartilage tissue injury is a meniscus injury, the collected tissue sample is preferably a meniscus cartilage tissue sample.

[0128] The cells contained in the tissue sample to be subjected to the method for enzymatic digestion of the present invention are preferably somatic cells such as primary somatic cells. More preferably, the cells are chondrocytes, cardiomyocytes, hepatocytes or pancreatic islet cells. Even more preferably, the cells are chondrocytes, with or without their pericellular matrix.

[0129] In the method for isolating the cells of the present invention, the yield of the isolated cells is preferably determined by counting. It is preferable to use a Bürker-Türk slide for yield measurement. For example, after sieving and washing the digest, the resulting cell suspension is repeatedly pipetted up and down to homogenize the concentration of the cells in the suspension. Next, a small sample is taken from the homogenized cell suspension and preferably mixed with a dye solution such as trypan blue. From this diluted suspension, 10 μL of the sample is taken twice to fill each of the two counting chambers of a Bürker-Türk slide. The cells are counted for each of the two chambers. By using the average count value together with the trypan blue dilution factor to calculate the cell concentration in the chamber, it becomes possible to calculate the cell concentration in the original sample. This concentration is assumed to represent the homogenized cell suspension.

[0130] The enzymatic digestion method of the present invention can be further used for other applications where the disassembly or degradation of the ECM is beneficial, such as meat tenderization or fish boning. Tenderization makes the meat more difficult to chew, more suitable, comfortable or acceptable for human consumption. In meat tenderization, the components of the extracellular matrix are partially degraded. Collagen is the main component that needs to be degraded to make the meat softer. The embodiments disclosed herein in relation to the method for enzymatic digestion of the present invention are also applicable to the method for tenderizing the meat products of the present invention.

[0131] The method for enzymatic digestion of the present invention can also be used for boning fish. Fish bones adhere to the fillets and cannot be easily removed before cooking. In the enzymatic boning of fish, the components of the extracellular matrix are partially decomposed. Collagen is a main component of fish bones and is one of the components that attach fish bones to the fillets. The embodiments disclosed herein in connection with the method for enzymatic digestion of the present invention are also applicable to the method for softening meat products of the present invention.

[0132] The present invention also provides a method for producing a peptide or a protein hydrolysate. The method includes the step of preparing an aqueous medium disclosed herein. The method also includes the step of contacting the aqueous medium with a composition containing a protein under conditions that enable enzymatic digestion of the protein.

[0133] Preferably, the peptide produced by the method is a bioactive peptide such as a collagen peptide, and the protein used in the method is collagen.

[0134] Preferably, the protein hydrolysate produced by the method is a collagen hydrolysate, and the protein used in the method is collagen.

[0135] Preferably, the bioactive peptide and / or the collagen hydrolysate consists of 2 to 100 amino acid residues. More preferably, the bioactive peptide and / or the collagen hydrolysate consists of 2 to 20 amino acid residues. Preferably, the bioactive peptide and / or the collagen hydrolysate is safe for human and / or animal consumption in a form such that normal use of the bioactive peptide or collagen hydrolysate by healthy non-allergic humans and / or animals does not cause serious diseases or illnesses.

[0136] Preferably, the bioactive peptide hydrolyzate is beneficial to the health of humans and / or animals. The bioactive peptide can, for example, affect the metabolism of humans and / or animals, prevent diseases, reduce chronic diseases, directly or indirectly cause a blood pressure lowering effect, directly or indirectly cause an antibacterial effect, directly or indirectly cause an antithrombotic effect, directly or indirectly cause an immunomodulatory effect, and / or directly or indirectly cause an effect on the mineral binding function.

[0137] Preferably, the bioactive peptide and / or the collagen hydrolyzate are used in the production of foods for human or animal consumption. Examples of such foods include formulated milk, infant nutrition, and protein powder.

[0138] Preferably, the bioactive peptide and / or the collagen hydrolyzate are produced from foods containing one or more proteins. The foods can include, for example, grains, milk, eggs, soybeans, fish, and meat.

[0139] Preferably, the bioactive peptide and / or the collagen hydrolyzate are the result of the hydrolysis of the one or more proteins.

[0140] The aqueous media disclosed herein can also find use in therapy. The use of collagenase in the treatment of disorders associated with the pathological accumulation of the extracellular matrix is well established. For example, the administration of collagenase is commonly used in wound debridement. Other indications in which collagenase is used and which are part of the present invention are the treatment of disorders including (skin) burns, keloid disease or placental remnants, mastodynia and intervertebral disc herniation, keloids, cellulite, lipomas, Peyronie's disease and Dupuytren's disease. Considering the improved enzymatic digestion of ECM proteins using the aqueous compositions as shown herein, it is also contemplated that the aqueous compositions of the present invention can improve ECM protein degradation in disorders associated with the pathological accumulation of the extracellular matrix, particularly in disorders characterized by the pathological accumulation of the extracellular matrix within or on the skin, or in body openings or the cavities associated therewith. Preferably, in such treatment methods, when the treatment of skin disorders such as wounds (e.g., non-healing or necrotic wounds), keloid disease, Peyronie's disease and Dupuytren's disease is contemplated, the aqueous compositions disclosed herein are applied topically. When the pathological accumulation of the ECM occurs in deeper skin layers, injection may be used, such as in the treatment of subjects with retained placenta, intraplacental injection may be used, etc. When the treatment of disorders in which the pathological accumulation of the ECM occurs in body cavities is contemplated, one of ordinary skill in the art can utilize other appropriate routes of administration. When the aqueous compositions disclosed herein are delivered into a body cavity, another appropriate route of administration may be administration by infusion.

[0141] A method for treating a subject having a cartilage tissue injury In another aspect, the method for enzymatic digestion of the present invention is part of a method for treating a subject having a cartilage tissue injury. The term "injury" can be used interchangeably with the term "defect" or "lesion".

[0142] In such a method, during a surgical procedure on a subject having cartilage tissue damage, a method for enzymatic digestion as disclosed herein is performed. In other words, such a method is performed during surgery and includes the steps of collecting or biopsying a cartilage tissue sample, such as a meniscus tissue sample, from a subject having cartilage tissue damage; optionally, mincing the tissue sample; performing a method for enzymatic digestion as disclosed herein; isolating chondrocytes and / or chondrons from the aqueous medium after enzymatic digestion; and re-seeding, transplanting, or implanting the isolated (autologous) chondrocytes and / or chondrons onto or into the cartilage tissue of the damaged subject, preferably at the site where the defect is located. Preferably, the collected cartilage tissue sample is obtained from an undamaged site in the damaged cartilage tissue.

[0143] In other words, the present invention provides a method for repairing a cartilage defect in a mammal in need thereof, the method comprising: a) providing an appropriate autologous tissue sample of chondrocytes and / or chondrons; b) performing a method for isolation as disclosed herein; c) optionally, mixing the chondrocytes and / or chondrons thus isolated with a matrix gel material; d) optionally, loading the chondrocytes and / or chondrons into or onto a scaffold; and e) transplanting the chondrocytes and / or chondrons or the loaded scaffold (optionally in the matrix gel) into the cartilage defect.

[0144] Numbered embodiments 1. Preparing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher; Contacting the aqueous medium with a tissue sample comprising an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein A method for enzymatic digestion of extracellular matrix proteins in a tissue sample, comprising: 2. The aqueous medium is Step of preparing an aqueous medium; Step of dissolving a calcium salt in the aqueous medium, where the final concentration of Ca 2+ in the aqueous medium is 2 mmol / L or more; and Step of mixing a matrix metalloproteinase with the aqueous medium before or after the dissolving step The method according to embodiment 1, prepared by a method comprising: 3. The method according to embodiment 1 or embodiment 2, wherein the step of contacting the aqueous medium with the tissue sample provides at least partial dissociation of the tissue sample, thereby releasing cells from the tissue sample into the aqueous medium. 4. The method according to any one of the preceding embodiments, wherein the method is for isolating cells with or without a pericellular matrix from a tissue sample. 5. The method according to any one of the preceding embodiments, wherein the method comprises isolating cells from the aqueous medium after enzymatic digestion, preferably by passing the digest through a cell strainer to isolate cells from the aqueous medium. The method according to any one of the preceding embodiments. 6. The method according to any one of the preceding embodiments, wherein the matrix metalloproteinase is selected from the group consisting of collagenase, gelatinase, stromelysin, matrilysin, metalloelastase, enamelysin, endometase and epilysin. 7. The method according to any one of the preceding embodiments, wherein the matrix metalloproteinase is collagenase. 8. The method according to any one of the preceding embodiments, wherein the aqueous medium comprises dissolved CaCl 2 . 9. The method according to any one of the preceding embodiments, wherein the tissue sample is selected from the group consisting of samples of cartilage tissue, muscle tissue, heart tissue, liver tissue, pancreatic tissue, thyroid tissue and salivary gland tissue. 10. The method according to any one of the embodiments, wherein the tissue sample is a collected or biopsied tissue sample, and optionally, a collected or biopsied tissue sample that is finely minced before the step of contacting the aqueous medium with the tissue sample. 11. The method according to any one of embodiments 3 to 10, wherein the cells are chondrocytes with or without their pericellular matrix, and the tissue sample is a cartilage tissue sample, preferably an articular cartilage tissue sample. 12. An aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher. 13. The aqueous medium according to embodiment 12, wherein the matrix metalloproteinase is collagenase. 14. The aqueous medium according to embodiment 12 or 13, wherein the aqueous medium further comprises an articular cartilage tissue sample and / or chondrocytes. 15. Use of the aqueous medium according to any one of embodiments 12 to 14 in extracellular matrix degradation, wherein the extracellular matrix comprises extracellular matrix proteins, preferably collagen. 16. The aqueous medium according to any one of embodiments 12 to 14 for use as a medicament. 17. The aqueous medium according to embodiment 16, which is for use in a method for treating a disorder associated with pathological accumulation of extracellular matrix, preferably pathological accumulation of extracellular matrix in the skin or on the skin or in body openings or the cavities associated therewith. 18. The aqueous medium according to embodiment 16, which is for use in a method for treating wounds, burns, keloid diseases, or placental remnants, or for use in wound debridement. 19. A step of preparing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher; contacting the aqueous medium with a meat product containing an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein A method for softening a meat product, comprising: 20. preparing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher; contacting the aqueous medium with a fish product containing an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein A method for boning a fish product, comprising: 21. preparing the aqueous medium according to any one of embodiments 12 to 14; contacting the aqueous medium with a composition containing a protein under conditions that allow enzymatic digestion of the protein A method for producing a peptide or a protein hydrolysate, comprising: 22. The method according to embodiment 21, wherein the peptide is a bioactive peptide such as a collagen peptide and the protein is collagen. 23. The method according to embodiment 21, wherein the protein hydrolysate is a collagen hydrolysate and the protein is collagen.

Example

[0145] Example 1. Isolation of chondrocytes from an articular cartilage tissue sample Materials and methods Fresh equine middle phalangeal joint or articular cartilage fragments were harvested from the middle phalangeal joint and stored at room temperature in RPMI (Roswell Park Memorial Institute) 1640 medium. A 2% collagenase solution was prepared by dissolving 200 mg of lyophilized collagenase powder (Collagenase Type 2, Worthington Biochemical Corporation, Lakewood, New Jersey, USA) in 10 ml of RPMI 1640. The solution was stored at 37 °C while the cartilage was minced. The cartilage fragments were manually minced with a scalpel, and a known amount of minced cartilage was transferred to a 12 ml tube together with 10 ml of the collagenase solution. In the control group, CaCl 2 was not added to this test tube. In the experimental group, 96 μl of 1 M CaCl 2 stock solution was added to the test tube to obtain a 10 mM Ca 2+ concentration. The tube was incubated at 37 °C for 35 minutes on a vortex shaker. The digest was gently resuspended with a 5 ml pipette and passed through a cell strainer with a pore size of 100 μm into a 50 ml tube. RPMI 1640 was added to the tube to fill it to 50 ml, and then it was centrifuged at 750 g for 2 minutes. The supernatant was discarded, the cell pellet was gently resuspended in 50 ml of RPMI 1640, and the tube was centrifuged at 750 g for 2 minutes. The supernatant was discarded again, the cell pellet was gently resuspended in a small amount of 1640 RPMI, and 15 μl of the sample was taken from it to count viable cells using a Bürker chamber. The results were expressed as isolated viable cells per gram of articular cartilage.

[0146] Results When equine articular cartilage was exposed to the collagenase solution while continuously shaking at 37 °C, 19,000 primary articular cartilage cells per gram of cartilage were harvested without Ca 2+ addition. At a Ca 2+ concentration of 10 mmol / L, 35,300 primary articular cartilage cells, which is approximately 1.9 times as many, were harvested per gram of cartilage (Figure 1).

[0147] Example 2. Harvesting Cartilage Cells from Bovine Articular Cartilage Materials and Methods The materials and methods were as described in Example 1. Articular cartilage fragments were harvested from fresh bovine middle finger joint or middle finger interphalangeal joint. One experimental group was added, and three groups were obtained: a control group, a 3 mM CaCl 2 group, and a 10 mM CaCl 2 group.

[0148] Results When bovine articular cartilage was exposed to the collagenase solution while continuously shaking at 37°C, 34,400 primary articular cartilage cells were harvested per gram of cartilage without Ca 2+ addition. At a Ca 2+ concentration of 3 mmol / L, 45,400 primary articular cartilage cells, which was about 1.3 times that of the control group, were harvested per gram of cartilage. When the Ca 2+ concentration in the digestive fluid was 10 mmol / L, 50,300 primary articular cartilage cells, which was about 1.5 times that of the control group, were harvested per gram of cartilage (Figure 2).

Claims

1. (i) preparing an aqueous medium comprising matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher; contacting the aqueous medium with a tissue sample containing extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein A method for enzymatic digestion of extracellular matrix protein in a tissue sample, comprising: The method, wherein the tissue sample is a cartilage tissue sample.

2. The aqueous medium is prepared by a method comprising: The step of dissolving a calcium salt in the aqueous medium, where the final concentration of Ca ２＋ in the aqueous medium is 2 mmol / L or more; and mixing matrix metalloproteinase with the aqueous medium before or after the dissolving step The method according to claim 1, prepared by a method comprising the steps.

3. The method according to claim 1 or claim 2, wherein the step of contacting the aqueous medium with the tissue sample provides at least partial dissociation of the tissue sample, thereby releasing cells from the tissue sample into the aqueous medium.

4. The method according to any one of claims 1 to 3, wherein the method is for isolating cells with or without a pericellular matrix from a tissue sample.

5. The method further comprises: isolating cells from the aqueous medium after enzymatic digestion, preferably by passing the digest through a cell strainer to isolate cells from the aqueous medium The method according to any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein the matrix metalloproteinase is selected from the group consisting of collagenase, gelatinase, stromelysin, matrilysin, metalloelastase, enamelysin, endometase and epilysin.

7. The method according to any one of claims 1 to 6, wherein the matrix metalloproteinase is collagenase, preferably the matrix metalloproteinase is limited to collagenase only, and more preferably, collagenase is the only protease present in the aqueous medium.

8. The aqueous medium contains dissolved CaCl ２ The method according to any one of claims 1 to 7, which contains

9. The method according to any one of claims 1 to 8, wherein the tissue sample is a collected or biopsied tissue sample, and optionally, the collected or biopsied tissue sample is minced before the step of contacting the aqueous medium with the tissue sample.

10. The method according to any one of claims 3 to 9, wherein the cell is a chondrocyte with or without its pericellular matrix, and preferably the tissue sample is an articular cartilage tissue sample.

11. An aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher.

12. The aqueous medium according to claim 11, wherein the matrix metalloproteinase is collagenase, preferably the matrix metalloproteinase is limited to collagenase only, and more preferably, collagenase is the only protease present in the aqueous medium.

13. The aqueous medium according to claim 11 or claim 12, wherein the aqueous medium further comprises an articular cartilage tissue sample and / or chondrocytes.

14. Use of the aqueous medium according to any one of claims 11 to 13 in extracellular matrix degradation, wherein the extracellular matrix comprises extracellular matrix proteins, preferably collagen.

15. The aqueous medium according to any one of claims 11 to 13 for use as a medicament.

16. An aqueous medium for use according to claim 15, wherein the aqueous medium is for use in a method of treating cartilage tissue damage.

17. An aqueous medium according to claim 15, wherein the aqueous medium is for use in a method of treating disorders associated with pathological accumulation of extracellular matrix, preferably pathological accumulation of extracellular matrix in or on the skin or at body openings or in the cavities associated therewith.

18. An aqueous medium according to claim 15, wherein the aqueous medium is for use in a method of treating wounds, burns, keloid diseases, or placental remnants, or for use in wound debridement.

19. A step of preparing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher; A step of contacting the aqueous medium with a meat product containing extracellular matrix proteins under conditions that allow enzymatic digestion of the extracellular matrix proteins A method for softening a meat product, comprising.

20. Preparing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation that is Ca2+ at a concentration of 2 mmol / L or higher; Contacting the aqueous medium with a fish product containing an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein A method for boning a fish product, comprising the steps above.

21. Preparing the aqueous medium according to any one of claims 11 to 13; Contacting the aqueous medium with a composition containing an extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein A method for producing a peptide or protein hydrolyzate, comprising the steps above.

22. The method according to claim 21, wherein the peptide is a bioactive peptide such as a collagen peptide and the protein is collagen.

23. The method according to claim 21, wherein the protein hydrolyzate is a collagen hydrolyzate and the protein is collagen.

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