Method for isolating cells from tissue sample

Abstract

The invention provides, inter alia, a method of enzymatic digestion of extracellular matrix proteins in a tissue sample, comprising the steps of:-providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) Ca2 + cations at a concentration of at least 2 mmol / L; -contacting said aqueous medium with a tissue sample comprising said extracellular matrix protein under conditions allowing the enzyme to digest said extracellular matrix protein; wherein the tissue sample is a cartilage tissue sample.

Description

Technical Field

[0001] The present invention relates to methods for enzymatic digestion of extracellular matrix (ECM) proteins. The in vitro or ex vivo methods described herein allow tissue dissociation and release of cells from the tissue, which can then be isolated and formulated for medical methods. The in vitro or ex vivo enzymatic digestion methods of the present invention improve the yield of isolated cells per unit tissue mass. The present invention also relates to aqueous media comprising matrix metalloproteinases, which can be used for applications in which extracellular matrix decomposition is beneficial, such as treatment of diseases associated with pathological accumulation of extracellular matrix, preferably pathological accumulation of extracellular matrix in or on the skin or in body orifices and their associated cavities. As disclosed herein, other applications that benefit from the use of aqueous media for extracellular matrix degradation are, for example, meat tenderization and fish deboning. Background Art

[0002] The use of cell-based medicine has increased over the past few decades. Cell-based medicine covers different fields such as cell transplantation, regenerative medicine, and tissue engineering. Examples of cell-based medicine include cardiac cell patches to repair cardiac lesions, kidney organoids to restore kidney function, pancreatic organoids to restore pancreatic function, intralesional injection of stromal cells from adipose or bone marrow to initiate or achieve a repair response, etc. The commonality of these cell-based therapeutic strategies is that they require the presence of sufficient numbers of tissue-isolated cells. Autologous cells are often used in cell-based medical approaches, where cells are harvested from the patient's own tissue to avoid immune rejection. The isolation of cells can be performed in a variety of ways, but may involve disrupting the extracellular matrix of the harvested tissue sample by enzymatic digestion with matrix metalloproteinases (such as collagenase) to dissociate the tissue and release the cells contained in the extracellular matrix.

[0003] An example of the tissue of interest relevant to cell-based medicine is cartilage tissue. Cartilage tissue comprises chondrocytes embedded in the extracellular matrix. In healthy cartilage tissue, chondrocytes produce and maintain the components of the extracellular matrix, such as collagen fibers, proteoglycans, glycosaminoglycans and elastin. Cartilage tissue may be damaged due to chondrodysplasia, arthritis, articular cartilage trauma and meniscus injury. Cartilage tissue can be repaired by autologous chondrocytes being transplanted into the cartilage tissue focus. For the autologous transplantation of chondrocytes, it is necessary to first separate chondrocytes from the cartilage tissue sample of the object, such as by enzymatic digestion to degrade or decompose the extracellular matrix in the tissue sample, to release the chondrocytes. Alternatively, less cartilage focus can be processed with ECM degrading enzyme solution, which can cause the permeability of ECM to increase, thereby increasing the migration potential of the chondrocytes residing around the focus.

[0004] Matrix metalloproteinases, also known as matrix metallopeptidases, are a class of endoproteases that degrade extracellular matrix proteins by hydrolysis of peptide bonds, including several types: collagen, gelatin, elastin, fibronectin, and laminin. One particularly prominent class of matrix metalloproteinases is collagenase. It is well known that collagenase is 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 described Ca 2+ Effects of Ca on conformational changes of Clostridial collagenase 2+ The concentration is far below 2mmol / L.

[0006] Ohbayashi et al., Appl Environ Microbiol. 2012; 78(16): 5839-5844 described Ca 2+ Effects on the structural and thermal stability of the type II collagenase Clostridial ColH. However, no Ca 2+ Effects on ECM digestion efficiency.

[0007] WO 2008 / 026928A1 describes a cartilage repair implant and mentions the content related to the separation of chondrocytes, that is, before subjecting the tissue sample to digestive enzymes, the tissue sample is treated to increase the permeability of the extracellular matrix, for example, by exposing the tissue sample to acid, alkali, dimethyl sulfoxide (DMSO), cathepsin, glycerol or cations (including Na + , K + NH4 + , pb 2+ Mg 2 + 、Zn 2+ , Fe 2+ 、Cd 2+ and Cu 2+ ) contact. For example, these cations can be introduced in the form of chloride salts at a concentration between 10 mM and 2 M. This suggests that after the permeability is increased, the tissue sample is washed before being subjected to the digestive enzyme treatment.

[0008] Although collagenase is known to contain zinc and requires calcium for activity (Khokha and Denhardt, Invasion Metastasis. 1989; 9(6): 391-405), the role of Ca in aqueous digestion media has not been studied to date. 2+ Effects of supraphysiological concentrations (i.e., concentrations of at least 2 mmol / L) of collagenase on the efficacy of collagenase-based ECM digestion.

[0009] There is a need in the art for further cell isolation methods, particularly methods that allow for high yield isolation of cells from tissue samples containing ECM. There is also a need for aqueous media containing digestive enzymes that can be used in therapeutic and non-therapeutic methods where the breakdown or degradation of the ECM is beneficial. Summary of the invention

[0010] The present inventors have unexpectedly determined that supraphysiological concentrations of calcium ions of at least 2 mmol / L can improve the yield of isolated cells per tissue sample when matrix metalloproteinases are incubated with tissue samples in aqueous media to break down ECM and release cells from the tissue samples.

[0011] More specifically, the present inventors surprisingly discovered that simultaneous exposure of cartilage tissue samples to matrix metalloproteinases and supraphysiological concentrations of calcium ions of at least 2 mmol / L in an aqueous medium can increase the yield of isolated cells per cartilage tissue sample.

[0012] Therefore, the present invention provides a method for enzymatic digestion of extracellular matrix proteins in a tissue sample, comprising the following steps: - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca(2+) solution having a concentration of at least 2 mmol / L; 2+ cations; - contacting the aqueous medium with a tissue sample comprising the extracellular matrix protein under conditions that allow enzymatic digestion of the extracellular matrix protein; wherein the tissue sample is a cartilage tissue sample.

[0013] In a preferred embodiment of the enzymatic digestion method, the aqueous medium is prepared by a method comprising the following steps: - providing an aqueous medium; - dissolving a calcium salt in the aqueous medium, wherein the Ca 2+ The final concentration of the matrix metalloproteinase is at least 2 mmol / L; and - before or after the solubilization step, mixing the matrix metalloproteinase with the aqueous medium.

[0014] In another preferred embodiment of the enzymatic digestion method, 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 enzymatic digestion method, the method is a method for isolating cells from a tissue sample, with or without their pericellular matrix.

[0016] In another preferred embodiment of the enzymatic digestion method, the method further comprises the following step: - separating cells from the aqueous medium after enzymatic digestion, preferably by passing the digestate through a cell strainer.

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

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

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

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

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

[0022] In another preferred embodiment of the enzymatic digestion method, the aqueous medium contains a cation, which is a supraphysiological concentration of Ca2+ higher than 10 mmol / L, such as higher than 15 mmol / L. 2+ .

[0023] In another preferred embodiment of the enzymatic digestion method, the aqueous medium contains dissolved CaCl2.

[0024] In another preferred embodiment of the enzymatic digestion method, the method does not include a step of incubating the tissue sample separately in a solution (such as a cationic solution) for increasing extracellular matrix permeability and / or a step of washing the tissue sample separately before incubating the tissue sample in an aqueous medium of the present invention. It has been determined that there is no need to perform a separate incubation step of the tissue sample in a solution (such as a cationic solution) for increasing extracellular matrix permeability in order to obtain a beneficial cell yield, and / or there is no need to perform a separate washing step before incubating the tissue sample in an aqueous medium of the present invention.

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

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

[0027] In another preferred embodiment of the enzymatic digestion method, the cell is a chondrocyte, with or without its 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, such as 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) Ca at a concentration of at least 2 mmol / L. 2+ cation.

[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 one matrix metalloproteinase selected from the group consisting of collagenase, gelatinase, stromelysin, matrix lytic protein, metalloelastase, enamelysin, endometase and epilysin.

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

[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 a supraphysiological concentration of Ca2+ higher than 10 mmol / L, such as higher than 15 mmol / L. 2+ .

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

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

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

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

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

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

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

[0041] In another aspect, the present invention provides an aqueous medium according to 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 used 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 used in a method for treating a condition associated with a pathological accumulation of an extracellular matrix, preferably a pathological accumulation of an extracellular matrix in or on the skin or in a body orifice or its associated cavities.

[0044] In another preferred embodiment of the aqueous medium for use as a medicament, the aqueous medium is used in a method for treating wounds, burns, keloid diseases or retained placenta, or for wound debridement.

[0045] In the same manner, the present invention provides a method for treating a condition associated with a pathological accumulation of an extracellular matrix, preferably a pathological accumulation of an extracellular matrix in or on the skin or in a body orifice or a cavity associated therewith, the method comprising the following steps: - administering to a subject in need thereof a therapeutically effective amount of an aqueous medium according to the present invention.

[0046] In a preferred embodiment of the method of treatment, the method is a method for treating wounds, burns, keloid disease or retained placenta, or for wound debridement.

[0047] In the same manner, the present invention provides the use of an aqueous medium of the invention for the preparation of a medicament for treating a condition associated with a pathological accumulation of extracellular matrix, preferably in or on the skin or in a body orifice or its associated cavities.

[0048] In the preferred embodiment using an aqueous medium, the method is a method for treating wounds, burns, keloid disease or retained placenta, or for wound debridement.

[0049] In another aspect, the present invention provides a method for tenderizing a meat product, comprising the steps of: - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca(II) solution having a concentration of at least 2 mmol / L; 2+ cations; - contacting the aqueous medium with a meat product comprising the 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: - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca2+ concentration of at least 2 mmol / L 2+ cations; - contacting the aqueous medium with the fish product comprising the 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 following steps: - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a cation, preferably a monovalent or divalent atomic cation; wherein the cation is at a supraphysiological concentration; - contacting the aqueous medium with a tissue sample comprising the 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) may be enhanced in swollen tissue samples. Swelling of tissue samples, such as cartilage tissue having many negatively charged components (such as negatively charged macromolecules), may be promoted by applying supraphysiological concentrations of calcium ions simultaneously with matrix metalloproteinases, which may result in increased osmotic pressure and water influx into the tissue sample.

[0053] The present invention also provides a method for treating cartilage tissue damage in a subject, such as meniscus damage (such as torn meniscus), the method comprising the following steps: - collecting or biopsy a cartilage tissue sample, such as a meniscus tissue sample, from a subject with cartilage tissue damage (such as meniscus damage); - optionally mincing the tissue sample; - implementing the enzymatic digestion method disclosed herein; isolating chondrocytes and / or cartilage units from the aqueous medium after enzymatic digestion; re-inoculating or transplanting the isolated (autologous) chondrocytes and / or cartilage units onto or in the injured cartilage tissue of the subject, for example, in or on the cartilage tissue lesion of the injured cartilage tissue.

[0054] In the same manner, the present invention provides an aqueous medium of the present invention, or a chondrocyte unit and / or chondrocyte population isolated from the aqueous medium, for use in a method for treating a subject with cartilage tissue damage, the method comprising the following steps: - applying, reseeding or transplanting (i) the aqueous medium comprising chondrocytes and / or chondrocyte units disclosed herein (preferably wherein the aqueous medium is a digest that has been passed through a filter disclosed herein), or (ii) the isolated (autologous) chondrocyte unit and / or chondrocyte population, to the damaged cartilage tissue of the subject; on or in it, for example in or on the cartilage tissue lesion (site) of the injured cartilage tissue; preferably, the separated cartilage units and / or chondrocyte populations can be obtained by a method comprising the following steps: - providing (or collecting or biopsy) a cartilage tissue sample, such as a meniscus tissue sample, obtained from a subject suffering from cartilage tissue injury (such as meniscus injury (such as a torn meniscus)); - optionally mincing the tissue sample; - implementing the enzymatic digestion method of the present invention; isolating chondrocytes and / or cartilage units from the aqueous medium after enzymatic digestion.

[0055] In another aspect, the present invention provides a method for producing a peptide or protein hydrolysate, comprising the steps of: - providing an aqueous medium of the present invention; and - contacting the aqueous medium with a composition comprising the protein under conditions allowing 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 hydrolysate is a collagen hydrolysate, and the protein is collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 . Yield of primary chondrocytes isolated from horse cartilage by enzymatic digestion (unit: million / g (10 6 / g)).

[0059] The control condition contained a calcium ion concentration of 0.423 mM. The chondrocyte yield under the control condition was 1.9 × 10 6 g -1 When the calcium ion concentration in the digestion medium was 10 mM, the chondrocyte yield was 3.5×10 6 g -1 .

[0060] Figure 2 . Yield of primary chondrocytes isolated from bovine cartilage by enzymatic digestion (unit: million / g (10 6 / g)).

[0061] The control condition contained a calcium ion concentration of 0.423 mM. The chondrocyte yield under the control condition was 3.4 × 10 6 g -1 When the calcium ion concentration in the digestion medium was 3 mM, the chondrocyte yield was 4.5 × 10 6 g -1 When the calcium concentration in the digestion medium was 10 mM, the chondrocyte yield was 5.0 × 10 6 g -1 . DETAILED DESCRIPTION

[0062] definition

[0063] As used herein, the terms "a" or "an", for example in relation to a matrix metalloprotease, include reference to one or more than one matrix metalloprotease.

[0064] As used herein, the term "enzymatic digestion" includes chemical transformation or subdivision of macromolecules by enzymatic activity. Preferably, the term relates to proteolytic cleavage of peptide bonds in proteins by enzymes such as matrix metalloproteinases. Preferably, the enzymatic digestion disclosed herein relates to the hydrolysis of proteins.

[0065] As used herein, the term "extracellular matrix" includes a network of macromolecules and minerals that provide structural support and / or protection to cells directly or indirectly nearby and interact with cells through cell surface receptors. The extracellular matrix includes interstitial matrix and basement membrane. The main components of animal extracellular matrix include proteoglycans, non-proteoglycan polysaccharides, extracellular vesicles and extracellular matrix proteins. The extracellular matrix can embed various cell types according to tissue type.

[0066] The term "extracellular matrix protein" as used herein includes proteins that are part of the extracellular matrix. In animals, the extracellular matrix proteins include collagen, elastin, fibronectin, laminin, actin and integrin, etc. 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.

[0067] As used herein, the term "tissue" includes reference to cellular organization levels between cells and complete organs. The term includes reference to cells and their extracellular matrix, which together perform a specific function. Non-limiting examples of tissues include connective tissue, epithelial tissue, muscle tissue, and neural tissue.

[0068] As used herein, the term "tissue sample" includes a tissue obtained from a subject, such as a tissue obtained by harvesting or as a biopsy. Tissue samples can be obtained from animal subjects (e.g., human subjects). Tissue samples are typically tissue portions, wherein the portions include cells and their extracellular matrix. It will be appreciated by those skilled in the art that if cells need to be separated, such as for subsequent autologous or allogeneic transplantation, the enzymatic digestion methods disclosed herein can be performed in vitro or ex vivo. Alternatively, the aqueous medium disclosed herein can be used to treat diseases associated with pathological accumulation of extracellular matrix. The term also includes any other non-animal tissue containing an extracellular matrix.

[0069] The term "incubation" as used herein includes actively or passively promoting contact between two or more chemical substances, mixtures, solutions, suspensions, emulsions, extractants, crystals, sols, colloids, gels, biomaterials, tissues, media or combinations thereof over a period of time. It is not intended to limit the length of the time period. External conditions, such as temperature, pH and agitation, may be part of the incubation process. Incubation allows reactions such as enzymatic digestion reactions to occur. Incubation can be carried out with or without an incubator container. When the term "incubation" used herein relates to the incubation of an aqueous medium disclosed herein with a tissue or tissue sample, it can be used interchangeably with the phrase "contacting the aqueous medium and tissue sample under conditions that allow enzymatic digestion of the extracellular matrix protein." It is clear to those skilled in the art the appropriate conditions that allow enzymatic digestion to occur.

[0070] As used herein, the term "aqueous medium" includes and refers to a liquid water-based medium, such as a liquid medium containing water as a major component. The medium may also include salts, anions, cations, carbohydrates, macromolecules, 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, a suspension or a colloid.

[0071] As used herein, the term "cation" includes meaning an ion with a positive charge. The cation can be, for example, a monovalent or divalent atomic ion, such as a monovalent or divalent metal ion. Preferably, the cation is a divalent metal ion, such as Ca 2+ Other cations that can be used in the present invention are Na + , K + NH4 + , Pb 2+ Mg 2+ , Mn 2+ 、Zn 2+ , Fe 2+ and Cu 2+ or Co 2+ 、Se 4+ or Cr 3+ .

[0072] The term "concentration" as used herein includes meaning the molar concentration (mol / L) of a compound in an aqueous medium. In the context of cations in an aqueous medium, the concentration refers to the concentration of the cation before the step of contacting the aqueous medium with a tissue sample, meat product or fish product disclosed herein.

[0073] As used herein, the term "matrix metalloproteinase (MMP)" includes a group of endoproteases that hydrolyze extracellular matrix proteins, whose function depends on the presence of at least one metal (such as calcium). Matrix metalloproteinases (MMPs) are also known as matrix metallopeptidases or matrixins. Preferably, the MMP is a collagenase, gelatinase, stromelysin, matrix cleavage protein (matrilysin), metalloelastase, enamelysin, endometase or epilysin. The group of matrix metalloproteinases includes gene products of 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 of their animals, plants, fungi, other eukaryotic organisms, bacteria, archaea and viruses homologs, orthologs, paralogs and counterparts. More preferably, the matrix metalloproteinase is a collagenase, such as MMP1, MMP2, MMP8, MMP9 or MMP13.

[0074] The term "mixing" as used herein in relation to the mixing of an MMP with an aqueous medium includes the meaning of mixing the MMP (which may be provided in solid form, for example in the form of a lyophilized, non-sterile powder, or pre-dissolved in a liquid medium) with the aqueous medium, for example by adding the MMP in solid or dissolved form to the aqueous medium while gently stirring.

[0075] The term "dissociation" as used herein refers to the dissociation of a tissue sample after enzymatic digestion, including at least partial loss of the integrity of the tissue sample caused by the enzymatic digestion of ECM proteins in the tissue sample, and the release of cells from the tissue sample. Cells released from the tissue sample are often no longer contained in the tissue sample, but may still be combined with other cells and / or other components that were previously part of the tissue, such as their pericellular matrix. An example of tissue sample dissociation is that a cartilage tissue sample is at least partially dissociated from the cartilage tissue sample during enzymatic digestion and release of chondrocytes, which may or may not have a pericellular matrix. The term "cell" as used herein includes cells that have or do not have a pericellular matrix. Preferred cells are chondrocytes or cartilage units. A cartilage unit refers to a chondrocyte with a pericellular matrix. If chondrocytes are mentioned herein, then cartilage units are included.

[0076] As used herein, the term "collagenase" includes an enzyme in the matrix metalloproteinase (MMP) group. It is known that collagenases can destroy the peptide bonds of collagen and procollagen, but are not limited thereto. Preferably, collagenases destroy peptide bonds by hydrolysis. Collagenases include other interstitial collagenases, neutrophil collagenases, collagenase 3, collagenase 4, clawed frog collagenases, fibroblast collagenases, PMN collagenases, type IV collagenases, ColH, ColG, AUX-I and AUX-II. The collagenases disclosed herein can be of animal, plant, fungal, bacterial or archaeal origin and can be used in the method of the present invention in a purified, partially purified or non-purified form. The collagenase used in the method disclosed herein can be commercially or non-commercially produced. The potential substrate of collagenase comprises any type of collagen, comprises I type collagen, II type collagen, III type collagen, IV type collagen and V type collagen, VI type collagen, IX type collagen, X type collagen, XI type collagen, XIIA type collagen, XIIB type collagen, XIV type collagen, XVI type collagen, XXII type collagen and XXVI type collagen.Another non-limiting example of potential substrate of collagenase is gelatin.Preferably, collagenase is microbial collagenase, preferably bacterial collagenase.

[0077] The term "gelatinase" as used herein includes an enzyme in the matrix metalloproteinase (MMP) group. Gelatinase can destroy the peptide bonds of collagen, procollagen and gelatin, but is not limited to this. Gelatinases include gelatinase A and gelatinase B, etc. Gelatinase can be of animal, plant, fungal, bacterial or archaeal origin, and can be used in a purified, partially purified or non-purified form. The gelatinase used in the method of the present invention can be commercially or non-commercially produced. Non-limiting examples of gelatinase substrates 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.

[0078] As used herein, the term "stromelysin" includes an enzyme that refers to the group of matrix metalloproteinases (MMPs). Stromelysins are capable of destroying the peptide bonds of collagen and procollagen, but are not limited thereto. Stromelysins include stromelysin-1, stromelysin-2, stromelysin-3, and stromelysin-4 (also referred to as RASI-1). Stromelysins can be of animal, plant, fungal, bacterial, or archaeal origin and can be used in a purified, partially purified, or non-purified form. The stromelysin used in the method of the present invention can be commercial or non-commercially produced. Non-limiting examples of stromelysin substrates include type II collagen, type IV collagen, type IX collagen, type X collagen, type XI collagen, gelatin, laminin, fibronectin, elastin, and aggrecan.

[0079] The term "matrilysin" as used herein includes an enzyme in the matrix metalloproteinase (MMP) group. Matrix lytic proteins can destroy the peptide bonds of collagen and procollagen, but are not limited thereto. Matrix lytic proteins include matrix lytic protein 1 (also known as PUMP-1 or uterine metalloproteinase) and matrix lytic protein 2 (also known as endometase). Matrix lytic proteins can be derived from animals, plants, fungi, bacteria or archaea, and can be used in a purified, partially purified or non-purified form. The matrix lytic proteins used in the method of the present invention can be commercial or non-commercially produced. Non-limiting examples of matrix lytic protein substrates 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, proteoglycans, fibrinogen, casein and vitronectin.

[0080] As used herein, the term "metalloelastase" and the expression "macrophage metalloelastase" include reference to an enzyme in the group of matrix metalloproteinases. Metalloelastases are capable of breaking peptide bonds in elastin, but are not limited thereto. Metalloelastases may be of animal, plant, fungal, bacterial or archaeal origin and may be used in purified, partially purified or non-purified form. The metalloelastase used in the method of the present invention may be produced commercially or non-commercially. Non-limiting examples of substrates for metalloelastases include elastin, type IV collagen, type I gelatin, fibronectin, laminin, vitronectin and proteoglycans.

[0081] As used herein, the term "enamelysin" includes an enzyme in the group of matrix metalloproteinases (MMPs). Anamelysin is capable of breaking peptide bonds in enamelin, but is not limited thereto. Anamelysin may be of animal, plant, fungal, bacterial or archaeal origin and may be used in a purified, partially purified or non-purified form. The anamelysin used in the method of the present invention may be produced commercially or non-commercially. Non-limiting examples of substrates for anamelysin include enamelin and amelogenin.

[0082] As used herein, the term "epilysin" includes a protein in the group of matrix metalloproteinases (MMPs). Epilysin is capable of breaking peptide bonds in casein, but is not limited thereto. Epilysin may be of animal, plant, fungal, bacterial or archaeal origin and may be used in a purified, partially purified or non-purified form. The epilysin used in the method of the present invention may be produced commercially or non-commercially.

[0083] As used herein, the term "dissolved" or "dissolving" includes meaning to form a solution comprising at least one solvent and at least one solute.

[0084] The terms "cartilage tissue" or "cartilaginous tissue" are used interchangeably and include tissue that refers to cartilage. Cartilage tissue can include tissue from elastic cartilage, hyaline cartilage and / or fibrous cartilage types. Preferably, the cartilage tissue is hyaline cartilage tissue. Cartilage tissue may be derived from any type of animal cartilage, including but not limited to joints, rib cages, ears, noses, bronchi and intervertebral discs. Cartilage tissue contains chondrocytes and an extracellular matrix in which the chondrocytes are embedded. The biochemical composition of cartilage tissue depends on its location (if applicable, the location before 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, 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.

[0085] Cartilage, preferably hyaline cartilage, comprises negatively charged components such as chondroitin sulfate-4, chondroitin sulfate-6, heparan sulfate and keratan sulfate. The architecture and composition of the extracellular matrix of cartilage are significantly different from other tissues such as pancreatic tissue and muscle tissue. The extracellular matrix of cartilage contains significantly more type II collagen and glycosaminoglycans (GAGs) than pancreatic tissue and / or muscle tissue. In addition, the extracellular matrix of cartilage is denser and tighter than, for example, pancreatic tissue and / or muscle tissue. The extracellular matrix represents the main functional component of cartilage, while cells represent the main functional components of other tissues, such as pancreatic tissue and / or muscle tissue.

[0086] As used herein, the term "cardiac tissue" includes tissues that refer to 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 extracellular matrix. The significant components of the extracellular matrix of cardiac tissue can be type I collagen and type III collagen.

[0087] As used herein, the term "liver tissue" includes tissues that refer to the liver. Liver tissue can include hepatocytes, Kuppfer cells, hepatic stellate cells, and sinusoidal endothelial cells. Liver tissue preferably includes an extracellular matrix. The significant components of the extracellular matrix of liver tissue are typically type I collagen, type III collagen, type IV collagen, and type V collagen.

[0088] As used herein, the term "pancreatic tissue" includes tissues that refer to the pancreas. Pancreatic tissue can include alpha cells, beta cells, delta cells, epsilon cells, PP cells (also known as gamma cells) and / or pancreatic connective tissue cells. Pancreatic tissue preferably includes an extracellular matrix. The significant components of the pancreatic tissue extracellular matrix are type I collagen, type III collagen, type IV collagen, and type V collagen.

[0089] As used herein, the term "thyroid tissue" refers to tissue of the thyroid gland. The thyroid tissue may include follicular cells and / or parafollicular cells. The thyroid tissue preferably includes an extracellular matrix. The significant components of the extracellular matrix of thyroid tissue are type I collagen and type III collagen.

[0090] As used herein, the term "salivary gland tissue" includes tissue that refers to one of the salivary glands. Salivary gland tissue may include serous cells, mucous cells, acinar cells, ductal cells and / or myoepithelial cells. Thyroid tissue preferably includes an extracellular matrix. A significant component of the extracellular matrix of thyroid tissue is type I collagen.

[0091] As used herein, the terms "harvesting" or "biopsy" include and refer to obtaining a tissue sample from a subject by methods and means known in the art.

[0092] The term "subject" as used herein preferably relates to mammals, such as humans. Other subjects may be non-human primates, domestic animals, such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals, such as mice, rats, rabbits, guinea pigs, etc., 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 condition associated with pathological accumulation of an extracellular matrix, preferably a pathological accumulation of an extracellular matrix in or on the skin or in a body orifice or its associated cavity, such as a wound, burn, keloid disease or retained placenta, or (ii) a human suffering from a cartilage tissue injury as disclosed herein. Pathological accumulation of the extracellular matrix means, in particular, excessive growth or excessive deposition of ECM, which, if not treated in time, can lead to adverse health conditions.

[0093] As used herein, the term "mincing" or "minced" includes reference to the mechanical division of a tissue sample, such as by cutting a harvested or biopsied tissue sample with a scalpel. Those skilled in the art are aware of the well-known methods and means for mincing a tissue sample.

[0094] As used herein, the term "isolate" includes reference to separating cells from a tissue sample, including reference to releasing cells from a tissue sample as a result of applying the enzymatic digestion methods of the invention and / or subsequently retrieving cells from the digestate, for example by passing the digestate through a cell strainer.

[0095] The term "digestate" as used herein includes the composition obtained after the step of contacting an aqueous medium with a tissue sample comprising the extracellular matrix proteins disclosed herein, i.e., an aqueous composition comprising enzymatically digested ECM proteins, at least partially dissociated tissue samples and / or cells released from said tissue samples.

[0096] As used herein, the term "chondrocyte" includes cells that produce cartilage tissue components and / or maintain the extracellular matrix of cartilage. The term encompasses cartilage units, i.e., chondrocytes with a pericellular matrix.

[0097] The term "articular cartilage tissue" as used herein includes a specific type of cartilage tissue found on the surface of bones with synovial joints. Articular cartilage is an example of hyaline cartilage. Articular cartilage tissue can be obtained from any suitable source, such as a meniscus.

[0098] As used herein, the term "treatment" includes administering or applying an active agent or a composition comprising an active agent to a subject for the purpose of partially or completely reversing, alleviating and / or inhibiting the progression of, or preventing, a disease or its symptoms.

[0099] As used herein, the term "therapeutically effective amount" includes an amount that has a therapeutic effect or an amount required to produce a therapeutic effect on a subject. For example, the therapeutically effective amount of the aqueous composition of the present invention is the amount required to produce the desired therapeutic effect, which can be judged by clinical trial results, model animal studies and / or in vitro studies. The therapeutically effective amount may depend on several factors, including but not limited to the characteristics of the subject (e.g., height, weight, sex, age, and medication history) and the type of disease.

[0100] As used herein, the term "wound" includes reference to any type of skin tissue injury associated with pathological accumulation of extracellular matrix.

[0101] The term "burn" as used herein includes skin wounds caused by thermal, chemical or radiation exposure or abrasive friction. The injury may be superficial or may affect deeper layers of the skin and underlying tissue. Preferably, the burn is associated with pathological accumulation of extracellular matrix.

[0102] As used herein, the term "keloid disease" includes reference to any disease associated with overgrowth of scar tissue that may migrate beyond the boundaries of the original skin lesion or lesion.

[0103] As used herein, the term "retained placenta" includes conditions where the placenta is partially or completely retained in the uterus after (vaginal) delivery. Generally, the placenta is retained when it is not expelled within 30 minutes after birth. For example, retained placenta can be treated by intraplacental administration of an aqueous composition of the present invention or umbilical cord injection of an aqueous composition of the present invention.

[0104] As used herein, the term "wound debridement" includes reference to enzymatic wound debridement, which is a standard of care therapy for treating non-healing and necrotic wounds in subjects who are not candidates for surgical intervention. Preferably, the subject is a subject with a non-healing or necrotic wound, and optionally also a subject who is not candidates for surgical intervention. Collagenase has been described as a preferred enzymatic debridement product for subjects with wounds containing, for example, yellow slough and / or scabs containing bacteria. Preferably, the wound debridement is enzymatic wound debridement.

[0105] As used herein, the term "tenderization" includes processes that make meat products easier to chew or easier to cut. Generally, the breakdown or degradation of extracellular matrix proteins such as collagen in meat products provides more tender meat products. Meat tenderization methods include mechanical tenderization, enzyme tenderization, acid tenderization, salt solution tenderization, sodium bicarbonate tenderization and / or heat tenderization. Preferably, in the method of the present invention, tenderization is enzyme tenderization.

[0106] The term "meat product" as used herein includes meat from slaughtered animals or artificial meat suitable for or can be made into suitable human consumption. Meat products generally include muscle tissue and / or fat tissue. Meat products can be meat products of pigs, cattle, sheep, chickens, turkeys, goats, ducks and / or deer.

[0107] The term "deboning" as used herein includes the process of separating fish fillets from fish bones. Deboning of fish products can be performed before or after they are prepared for consumption. Preferably, in the method of the present invention, the fish product is a decapitated, gutted and / or finned fish, which may also be optionally tailed.

[0108] The term "fish product" as used herein includes and means a product comprising both fish meat (fish fillet) and bones. Preferably, the fish product is a decapitated, gutted and / or finned fish, which may also be optionally tailed.

[0109] As used herein, the phrase "body orifice or its associated cavity" includes meaning any body opening and its associated extracellular cavity. Examples of body orifices and their associated cavities include the nostrils and nasal cavity, the mouth and throat, the ear opening and ear canal, the anus and the intestines, such as the rectum, the urethral opening and urethra, and the bladder, vagina, and uterus.

[0110] As used herein, the term "supraphysiological concentration" includes cations (such as Ca 2+ ) concentration, wherein the concentration is higher than the concentration of the cation normally experienced by the tissue to be enzymatically digested. For example, for Ca 2+ , physiological concentrations in different tissue types do not exceed 2mmol / L. For other cations, it should be noted that the concentration range of trace elements in mammalian serum is iron 0.0179-0.0358mmol / L, zinc 0.0122-0.0184mmol / L, copper 0.00897-0.0157mmol / L, cobalt 0.170-0.509μmol / L, manganese 3.28-3.46μmol / L, selenium 0.633-2.79nmol / L (Yatoo et al., Veterinary World. 2013; 6(12): 963-967). The cation concentrations of these elements in the aqueous medium of the present invention exceeding these ranges (e.g., measured in serum) can be considered supraphysiological.

[0111] The term "bioactive peptide" as used herein includes peptides that preferably show beneficial effects on the human or animal body due to its biological activity. Bioactive peptides are generally derived from food, such as protein-containing materials from plants and / or animal products, including grains, milk, eggs, soybeans, fish and meat. Bioactive peptides are generally obtained by hydrolyzing whole proteins. The beneficial effects of bioactive peptides include, but are not limited to, affecting human or animal metabolism, preventing diseases, reducing chronic diseases, antihypertensive effects, antibacterial effects, antithrombotic effects, immunomodulatory effects and effects on mineral binding functions. Bioactive peptides can be produced by enzymatic methods, food processing or microbial fermentation. Preferably, the composition comprising the protein disclosed herein comprises collagen (i.e., an enzymatically digestible protein that can be enzymatically digested by the matrix metalloproteinase enzyme disclosed herein as a collagen peptide). In addition, the composition comprising the protein disclosed herein can be a whole protein preparation.

[0112] As used herein, the term "protein hydrolysate" includes and means whole protein after digestion, making it into protein fragments. Protein hydrolysates may be more easily and quickly absorbed by the human or animal body than whole proteins. Protein hydrolysates are also known to be associated with cell culture.

[0113] As used herein, the term "whole protein preparation" includes proteins that have not been completely digested, and preferably have not (yet) been partially digested. These proteins can form the basis for the production of biologically active peptides and / or protein hydrolysates.

[0114] Method for isolating cells from tissues by enzymatic digestion of ECM

[0115] Isolation of cells from tissue has been found to be challenging and does not always yield the desired number of cells. This applies particularly to tissue types where the presence of an extracellular matrix prevents the release and separation of cells from the tissue. An example of such a tissue is cartilage tissue, which contains large amounts of glycosaminoglycans, proteoglycans, collagen and elastin fibers. Isolation of cells from tissue is necessary for multicellular-based pharmaceutical applications. If the isolation process is inefficient, a larger tissue sample will need to be provided in order to yield a sufficient number of isolated cells.

[0116] The present invention now provides a method for treating a patient by using a cell culture medium comprising a matrix metalloproteinase and a supraphysiological concentration of a cation such as Ca 2+A method for efficiently separating cells from a tissue sample by enzymatic digestion of ECM proteins in an aqueous medium. This enzymatic digestion method is particularly useful for separating chondrocytes and / or cartilage units from cartilage tissue. Since the method of the present invention is particularly directed to improved digestion of extracellular matrix proteins in the extracellular matrix of a tissue sample by matrix metalloproteinases, it is also applicable to other processes requiring the decomposition of extracellular matrix proteins, such as treatment of wounds, burns, keloid diseases or retained placenta, enzymatic wound debridement, meat tenderization or fish deboning. The advantageous application of matrix metalloproteinases such as collagenase in these methods has been previously determined. For example, in enzymatic wound debridement, collagenase is a standard care therapy for non-healing and necrotic wounds in subjects who cannot choose surgical intervention.

[0117] Preferably, in the method of the present invention for enzymatic digestion or separation of cells from tissue, the first step comprises providing a tissue sample from a subject, for example by collection or as a biopsy. Such a subject may be a subject with cartilage tissue damage, such as an articular cartilage lesion. Suitable methods and means for providing a tissue sample are well known to those skilled in the art. In addition, this step of collecting a tissue sample may be intraoperative, i.e., may be part of a method for treating a subject with cartilage tissue damage (such as an articular cartilage lesion), as disclosed herein.

[0118] Prior to enzymatic digestion, the harvested or biopsied tissue is optionally minced to increase the surface area of ​​the tissue. Mincing can be performed, among other things, by cutting the harvested or biopsied tissue sample with any suitable instrument, such as a scalpel.

[0119] Subsequently, the tissue sample is treated with a mixture containing a matrix metalloproteinase and a supraphysiological concentration (i.e., compared to the normal physiological concentration of the cation in the tissue or serum) of at least one cation, such as Ca. 2+ After an appropriate incubation time, under appropriate incubation conditions, the resulting digest can be filtered through a cell strainer with a pore size of 100 μm, etc. to filter out undigested tissue. The filtered digest can be washed with an appropriate solution (such as phosphate-buffered saline) to elute or wash away the medium containing matrix metalloproteinases. The washed digest will be a suspension of individual and clustered primary cells containing varying amounts of residual extracellular matrix and extracellular matrix fragments, from which representative samples can be collected to assess the cell concentration of the digest. The terms "contact" and "incubation" are used interchangeably herein.

[0120] Ca 2+ The physiological concentration of Ca may vary depending on the organ and tissue type. For example, in mammals, the extracellular environment Ca 2 + The physiological concentration of Ca in human serum is 1-2mmol / L.2+ The concentration is, for example, 1.1 to 1.3 mmol / L. In human synovial fluid, Ca 2+ The concentration is 0.32mmol / L. Physiological concentration refers to the concentration at or in a tissue of interest in a living healthy animal (such as a human) under steady-state conditions. Preferably, the physiological concentration is the extracellular concentration of cations at or in a tissue of interest in a living healthy animal under steady-state conditions. Supraphysiological concentration refers to a concentration exceeding the physiological concentration at or in the tissue of interest. 2+ The concentration is not at least 2mmol / L, so supraphysiological Ca can be accepted 2+ The concentration is at least or higher than 2 mmol / L. In an embodiment, the aqueous medium preferably contains at least 2 mmol / L of Ca 2+ or Ca higher than 2mmol / L 2 + , more preferably between 2 and 2000mmol / L, more preferably between 2 and 200mmol / L, even more preferably between 2-50mmol / L, most preferably between 3 and 10mmol / L, such as 3, 4, 5, 6, 7, 8, 9 or 10mmol / L. The aqueous medium may contain at least 10mmol / L of Ca 2+ The aqueous medium may contain greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 and 50 mmol / L of Ca. 2+ The aqueous medium may contain 15-50, 15-40, 15-30 or 15-20 mmol / L of Ca 2+ . Cations such as Ca 2+ Any salt may be added to the aqueous medium, such as CaSO4, calcium citrate, CaCO3, CaBr2, CaF2, CaI2, CaCl2, Ca(NO3)2, CaC2O4, Ca(H2PO4)2 or CAHPO4. Most preferably, Ca 2+ The cation is added to the aqueous medium in the form of its CaCl2 salt.

[0121] In the methods of the present invention, enzymatic digestion is performed by one or more matrix metalloproteinases (MMPs), as disclosed herein, which are contained in an aqueous medium. Examples of MMPs are collagenases, gelatinases, stromelysins, matrix lysins, metalloelastases, enamelysins, endometases, and epilysins. MMPs used in the methods of the present invention include animal, plant, fungal, and bacterial MMPs and homologs, orthologs, paralogs, and counterparts thereof, such as the MEROPS peptidase family M9 enzyme. 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, M9B subfamily proteins from Microscilla marina, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, and the like. subtilis), Rhizoctonia solani, Thermoactinomyces E-21, and Clostridium perfringens. Preferably, the MMP disclosed herein is a collagenase. More than one type of collagenase may be used in the methods of the present invention. More preferably, type II collagenase is used. The amount of the MMP in the aqueous medium as disclosed herein is preferably between 0.2% w / v and 10% w / v. Preferably, the amount of the MMP in the aqueous medium is about 2% w / v.

[0122] Aqueous media as disclosed herein may also include a buffer to maintain a pH corresponding to physiological pH, such as a pH of about 7. The concentration of the buffer may be between 1 and 100 mM. Examples of suitable buffers include organic and inorganic compounds and salts thereof, such as citrate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, phosphate, carbonate, bicarbonate and borate buffers. In addition, aqueous media may include amino acid-based buffers, urea buffers and buffers such as Tris, MOPS, ACES, PIPES, BES and HEPES.

[0123] Other components that may be included in the aqueous medium disclosed in this article are DTPA, EDTA, EGTA; reducing agents such as dithiothreitol, dithioerythritol, β-mercaptoethanol, glutathione, thioredoxin, cysteine, ascorbic acid, thioglycolate; ions required for activating any other components in the medium, such as MgCl2, NaCl, KCl, ZnCl2, ZnSO4; organic solvents or lipid / membrane modifiers, such as DMSO; non-ionic detergents, such as Triton X-100; and / or osmoprotectants, such as sucrose.

[0124] In a preferred embodiment, the enzymatic digestion method of the present invention comprises the step of preparing an aqueous medium disclosed herein, comprising the following steps: - providing an aqueous medium; - dissolving a calcium salt in the aqueous medium, wherein the calcium salt in the aqueous medium is 2+ The final concentration is at least 2 mmol / L; and

[0125] - Before or after the solubilization step, the matrix metalloproteinase is mixed with the aqueous medium. The preparation can be carried out by adding the stock solution components together to reach the desired concentration and appropriately and thoroughly mixing the components. The mixing can be carried out by pipetting, shaking, stirring, vortexing or mixing, ultrasonic treatment and / or passive mixing. Preferably, the mixing is carried out by vortexing. Preferably, the mixing of the aqueous medium lasts for 0.1 to 20 minutes.

[0126] In the enzymatic digestion method of the present invention, the tissue sample containing the extracellular matrix protein is mixed with the aqueous medium disclosed herein. Mixing can be accomplished by blowing, shaking, stirring, vortexing or mixing, ultrasonic treatment and / or passive mixing. Preferably, the tissue sample and the medium are mixed by vortexing.

[0127] As described herein, incubating or contacting an aqueous medium with a tissue sample means that the tissue sample is at least partially, preferably completely, immersed in an aqueous medium. During the incubation process, the tissue sample contacts the aqueous medium and thereby contacts its components. During the incubation process, 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 the incubation process can be adjusted. Preferably, the temperature during the incubation period is constant. More preferably, the temperature during the incubation period is constant at about 37°C. Incubation can be carried out for 1 to 120 minutes. Preferably, incubation is carried out for 5 to 60 minutes. More preferably, incubation takes about 35 minutes.

[0128] In an embodiment, the method of the invention comprises the step of separating or isolating cells from said aqueous medium after enzymatic digestion, preferably by passing the digestate through a cell filter having a pore size that allows cells to pass through but blocks larger components. Preferably, the cell filter has a pore size of about 100 μm.

[0129] The tissue sample disclosed herein can be of any size or shape. Preferably, it is a fragment or subcomponent of a tissue containing ECM in an animal body. The tissue sample is preferably an animal tissue sample, more preferably a human tissue sample.

[0130] Preferably, the tissue sample disclosed herein is selected from the group consisting 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 consisting of cartilage tissue, heart tissue, liver tissue, pancreatic tissue, thyroid tissue, and salivary gland tissue.

[0131] Even more preferably, the tissue sample is a cartilage tissue sample. Cartilage tissue mainly has three types, i.e. 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 articular cartilage tissue.

[0132] The articular cartilage tissue disclosed herein can be harvested or biopsied from any suitable location in an animal. Examples of locations in an animal body suitable for harvesting cartilage tissue are, for example, metacarpophalangeal joints, metatarsophalangeal joints, interphalangeal joints, interphalangeal joints, intermetatarsal joints, tarsometatarsal joints, tarsometatarsal joints, intercuneiform joints, cuboideonavicular joints, cuboscaphoid joints, calcaneocuboid joints, talocalcaneal joints, talocrural joints, tibiofibular joints, patellofemoral joints, tibiofemoral joints, hip joints, sternoclavicular joints, acromioclavicular joints, glenohumeral joints, humeroradial joints, humeroulnar joints. The cartilage is collected from the metacarpophalangeal joint, the midcarpal joint, the carpometacarpal joint, the intercarpal joint, the interphalangeal joint, the atlantoaxial joint, the facet joint, the lumbosacral joint, the sacrococcygeal joint, the costovertebral joint, the sternocostal joint, the intercartilaginous joint, the costochondral joint, the pubic symphysis, the sacroiliac joint, the temporomandibular joint and the atlanto-occipital joint. Preferably, the cartilage is collected from the metacarpophalangeal joint and / or the metatarsophalangeal joint.

[0133] Preferably, in a method of treating a subject with cartilage tissue damage, the cartilage tissue sample is harvested from the same tissue as the tissue containing the cartilage tissue damage (also referred to as a defect or lesion / foci). For example, if the cartilage tissue damage is a meniscus damage, the harvested tissue sample is preferably a meniscus cartilage tissue sample.

[0134] The cells contained in the tissue sample subjected to the enzymatic digestion method 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.

[0135] In the method for separating cells of the present invention, the yield of separated cells is preferably determined by counting. Preferably, a Bürker-Türk slide is used for yield determination. For example, after filtering and washing the digest, the resulting cell suspension is repeatedly pipetted up and down to homogenize the concentration of cells in the suspension. Then, a small sample is taken from the homogenized cell suspension and preferably mixed with a dye solution such as trypan blue. From the diluted suspension, two 10-μL samples are taken to fill the two counting chambers of the Bürker-Türk slide. Cells are counted in each of the two chambers. The average count is used to calculate the cell concentration in the chamber, and together with the trypan blue dilution factor, the cell concentration in the original sample can be calculated. It is presumed that this concentration represents a homogenized cell suspension.

[0136] The enzymatic digestion method of the present invention can also be used for other applications where ECM breakdown or degradation is beneficial, such as meat tenderization or fish deboning. Tenderization makes meat less chewy and more suitable, more pleasant or easier for humans to eat. During the meat tenderization process, components of the extracellular matrix are partially broken down. Collagen is a major component that needs to be broken down to make the meat more tender. The embodiments disclosed herein related to the enzymatic digestion method of the present invention are also applicable to the method of tenderizing a meat product of the present invention.

[0137] The enzymatic digestion method of the present invention can also be used for deboning fish. Fish bones are attached to fish fillets and are not easily removed before cooking. During the enzymatic deboning of fish, components of the extracellular matrix are partially decomposed. Collagen is the main component of fish bones and is one of the components that attach fish bones to fish fillets. The embodiments disclosed herein related to the enzymatic digestion method of the present invention are also applicable to the method of tenderizing a meat product of the present invention.

[0138] The present invention also provides a method for producing peptide or protein hydrolysate. The method comprises the following steps: - providing an aqueous medium disclosed herein. The method also comprises the following steps: - contacting the aqueous medium with a composition comprising protein under conditions that allow enzyme digestion of the protein.

[0139] Preferably, the peptides produced by the method are bioactive peptides, such as collagen peptides, and the protein used in the method is collagen.

[0140] Preferably, the protein hydrolysate produced by the method is a collagen hydrolysate, and the protein used in the method is collagen.

[0141] 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, and normal use of such bioactive peptides or collagen hydrolysate by healthy, non-allergic humans and / or animals will not cause serious diseases.

[0142] Preferably, the bioactive peptide hydrolysate is beneficial to human and / or animal health. The bioactive peptides may, for example, affect human and / or animal metabolism, prevent disease, reduce chronic disease, directly or indirectly cause antihypertensive effects, directly or indirectly cause antibacterial effects, directly or indirectly cause antithrombotic effects, directly or indirectly cause immunomodulatory effects and / or directly or indirectly affect mineral binding function.

[0143] Preferably, the bioactive peptides and / or the collagen hydrolysate are used to produce food for human or animal consumption. Examples of the food include formula milk, infant nutrition and protein powder.

[0144] Preferably, the bioactive peptides and / or the collagen hydrolysate are produced from foods containing one or more proteins, such as cereals, milk, eggs, soybeans, fish and meat.

[0145] Preferably, the bioactive peptides and / or the collagen hydrolysate are the result of hydrolysis of the one or more proteins.

[0146] The aqueous medium disclosed herein can also be used for treatment. The use of collagenase in the treatment of diseases associated with pathological accumulation of extracellular matrix has been demonstrated. For example, administration of collagenase is generally used for wound debridement. Other indications for the use of collagenase, which are also part of the present invention, are the treatment of (skin) burns, keloid disease or retained placenta, nipple pain and conditions including herniated discs, keloids, cellulite, lipomas, Peyronie's disease and Dupuytren's contracture. In view of the improved enzymatic digestion of ECM proteins using the aqueous composition shown herein, it is also conceivable that the aqueous composition of the present invention allows for improved ECM protein degradation in diseases associated with pathological accumulation of extracellular matrix, particularly those characterized by pathological accumulation of extracellular matrix in or on the skin or in body orifices or their associated cavities. Preferably, in this method of treatment, when it is envisaged to treat skin disorders such as wounds (e.g., non-healing wounds or necrotic wounds), keloid diseases, Peyronie's disease and Dupuytren's contracture, the aqueous composition disclosed herein is topically applied. When it is envisaged to treat diseases in which ECM accumulates pathologically in body cavities, other suitable routes of administration may be used by those skilled in the art, such as by injection in the case of pathological accumulation of ECM in deeper skin layers, or by intraplacental injection in the treatment of subjects with retained placentas. When the aqueous composition disclosed herein is to be delivered to a body cavity, other suitable routes of administration may be by instillation.

[0147] Methods of treating subjects with cartilage tissue damage

[0148] In another aspect, the enzymatic digestion method of the invention is part of a method of treating a subject with cartilage tissue damage.The term "damage" may be used interchangeably with the term "defect" or "lesion / foci".

[0149] In this method, the enzymatic digestion method disclosed herein is performed during surgical treatment of a subject with cartilage tissue damage. In other words, this method is performed during surgery and may include the following steps: - harvesting or biopsying a cartilage tissue sample, such as a meniscus tissue sample, from a subject with cartilage tissue damage (such as meniscus damage); - optionally mincing the tissue sample; - implementing the enzymatic digestion method disclosed herein; isolating chondrocytes and / or cartilage units from the aqueous medium after enzymatic digestion; re-seeding, transplanting or implanting the isolated (autologous) chondrocytes and / or cartilage units onto or in the cartilage tissue of the injured subject, preferably at the site of the defect. Preferably, the harvested cartilage tissue sample is obtained from injured cartilage tissue but at an uninjured site.

[0150] In other words, the present invention provides a method for repairing cartilage defects in a mammal in need thereof, comprising the following steps: a) providing a tissue sample of suitable autologous origin of chondrocytes and / or chondrocyte units; b) implementing the separation method disclosed herein; c) optionally, mixing the chondrocytes and / or chondrocyte units thus separated with a matrix gel material; d) optionally, loading the chondrocytes and / or chondrocyte units in or on a scaffold, and e) implanting the cells and / or chondrocyte units (optionally in the matrix gel) or the loaded scaffold into the cartilage defect.

[0151] Multiple Implementations

[0152] 1. A method for enzymatic digestion of extracellular matrix proteins in a tissue sample, comprising the following steps:

[0153] - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca2+ concentration of at least 2 mmol / L 2+ cation;

[0154] - contacting said aqueous medium with a tissue sample comprising extracellular matrix proteins under conditions allowing enzymatic digestion of said extracellular matrix proteins.

[0155] 2. The method of embodiment 1, wherein the aqueous medium is prepared by a method comprising the following steps:

[0156] -Providing an aqueous medium;

[0157] - dissolving a calcium salt in the aqueous medium, wherein the aqueous medium contains Ca 2+ The final concentration is at least 2 mmol / L; and

[0158] - before or after said solubilization step, mixing a matrix metalloproteinase with said aqueous medium.

[0159] 3. The method of embodiment 1 or 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 in the aqueous medium.

[0160] 4. The method according to any one of the preceding embodiments, wherein the method is a method for isolating cells from a tissue sample, with or without their pericellular matrix.

[0161] 5. The method according to any one of the preceding embodiments, wherein the method further comprises the following steps:

[0162] - After enzymatic digestion the cells are preferably separated from the aqueous medium by passing the digest through a cell strainer.

[0163] 6. The method of any of the preceding embodiments, wherein the matrix metalloproteinase is selected from the group consisting of collagenases, gelatinases, stromelysins, matrix lytic proteins, metalloelastases, amelolysins, matrix enzymes in the inner membrane, and epidermal hydrolases.

[0164] 7. The method of any one of the preceding embodiments, wherein the matrix metalloproteinase is collagenase.

[0165] 8. The method of any one of the preceding embodiments, wherein the aqueous medium comprises dissolved CaCl2.

[0166] 9. The method of 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.

[0167] 10. The method of any one of the preceding embodiments, wherein the tissue sample is a harvested or biopsied tissue sample, optionally a harvested or biopsied tissue sample that is minced prior to the step of contacting the aqueous medium with the tissue sample.

[0168] 11. The method of any one of embodiments 3-10, 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.

[0169] 12. An aqueous medium comprising (i) a matrix metalloproteinase and (ii) Ca at a concentration of at least 2 mmol / L. 2+ cation.

[0170] 13. The aqueous medium of embodiment 12, wherein the matrix metalloproteinase is collagenase.

[0171] 14. The aqueous medium of embodiment 12 or 13, wherein the aqueous medium further comprises an articular cartilage tissue sample and / or chondrocytes.

[0172] 15. Use of the aqueous medium as described in any one of embodiments 12-14 in the degradation of extracellular matrix; wherein the extracellular matrix comprises extracellular matrix proteins, preferably collagen.

[0173] 16. The aqueous medium of any one of embodiments 12-14 for use as a medicament.

[0174] 17. An aqueous medium for use according to embodiment 16, wherein the aqueous medium is used in a method of treating a condition associated with pathological accumulation of extracellular matrix, preferably pathological accumulation of extracellular matrix in or on the skin or in a body orifice or a cavity associated therewith.

[0175] 18. The aqueous medium for use according to embodiment 16, wherein the aqueous medium is used in a method for treating wounds, burns, keloid disease or retained placenta, or for wound debridement.

[0176] 19. A method for tenderizing a meat product, comprising the steps of:

[0177] - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca2+ concentration of at least 2 mmol / L 2+ cation;

[0178] - contacting said aqueous medium with a meat product comprising extracellular matrix proteins under conditions allowing enzymatic digestion of said extracellular matrix proteins.

[0179] 20. A method for deboning fish products comprising the steps of:

[0180] - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca2+ concentration of at least 2 mmol / L 2+ cation;

[0181] - contacting said aqueous medium with a fish product comprising extracellular matrix proteins under conditions allowing enzymatic digestion of said extracellular matrix proteins.

[0182] 21. A method for producing a peptide or protein hydrolysate, comprising the steps of:

[0183] - providing an aqueous medium as described in any one of embodiments 12-14;

[0184] - contacting said aqueous medium with a composition comprising said protein under conditions allowing enzymatic digestion of the protein.

[0185] 22. The method of embodiment 21, wherein the peptide is a bioactive peptide, such as a collagen peptide, and the protein is collagen.

[0186] 23. The method of embodiment 21, wherein the protein hydrolysate is a collagen hydrolysate, and the protein is collagen.

[0187] Example

[0188] Example 1. Isolation of chondrocytes from articular cartilage tissue samples.

[0189] Materials and methods

[0190] Articular cartilage blocks were collected from fresh metacarpophalangeal or metatarsophalangeal joints and stored in RPMI (Lossville Park Memorial Institute) 1640 medium at room temperature. A 2% collagenase solution was prepared by dissolving 200 mg of lyophilized collagenase powder (collagenase type 2, Worthington Biochemical Corporation, Lakewood, NJ, USA) in 10 ml RPMI 1640. The solution was stored at 37°C during cartilage mincing. The cartilage blocks were manually minced with a scalpel, and a known amount of minced cartilage was transferred to a 12-ml test tube containing 10 ml of collagenase solution. In the control group, CaCl2 was not added to the test tube. In the experimental group, 96 μl of 1 M CaCl2 stock solution was added to the test tube to make Ca 2+ The concentration is 10 mM. The test tubes are incubated on a vortex shaker for 35 minutes at 37°C. The digest is gently resuspended with a 5-ml pipette and passed through a cell strainer with a pore size of 100 μm into a 50 ml test tube. RPMI 1640 is added to the test tube, filling up to 50 ml, and then centrifuged at 750g for 2 minutes. The supernatant is discarded, the cell pellet is gently resuspended in 50 ml RPMI 1640, and the test tube is centrifuged again at 750g for 2 minutes. The supernatant is discarded again, and the cell pellet is gently resuspended in a small amount of 1640 RPMI, from which a 15 μl sample is taken and the viable cells are counted using a Bürker-Türk chamber. The results are expressed as viable cells isolated per gram of articular cartilage.

[0191] result

[0192] When horse articular cartilage was exposed to collagenase solution at 37°C under continuous shaking, without the addition of Ca 2+ 1.9 million primary articular chondrocytes were harvested per gram of cartilage. 2+ At a concentration of 10 mmol / L, 3.53 million primary articular chondrocytes were harvested per gram of cartilage; approximately 1.9 times ( Figure 1 ).

[0193] Example 2. Chondrocytes were collected from bovine articular cartilage.

[0194] Materials and methods

[0195] Materials and methods are as described in Example 1. Articular cartilage blocks were obtained from fresh bovine metacarpophalangeal joints or metatarsophalangeal joints. An experimental group was added, divided into three groups: control group, 3 mM CaCl2 and 10 mM CaCl2.

[0196] result

[0197] When bovine articular cartilage was exposed to collagenase solution at 37°C under continuous shaking, without the addition of Ca2+ When the digestate solution contained Ca 2+ When the concentration was 3 mmol / L, 4.54 million primary articular chondrocytes were harvested per gram of cartilage, which was approximately 1.3 times that of the control group. 2+ At a concentration of 10 mmol / L, 5.03 million primary articular chondrocytes were harvested per gram of cartilage, which is approximately 1.5 times that of the control group ( Figure 2 ).

Claims

1. A method for enzymatic digestion of extracellular matrix proteins in a tissue sample, comprising the following steps: - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca2+ concentration of at least 2 mmol / L 2+ cation; - contacting the aqueous medium with a tissue sample comprising the extracellular matrix proteins under conditions that allow enzymatic digestion of the extracellular matrix proteins; Wherein the tissue sample is a cartilage tissue sample.

2. The method of claim 1, wherein the aqueous medium is prepared by a method comprising the steps of: -Providing an aqueous medium; - dissolving a calcium salt in the aqueous medium, wherein the aqueous medium contains Ca 2+ The final concentration is at least 2 mmol / L; and - before or after said solubilization step, mixing a matrix metalloproteinase with said aqueous medium.

3. The method of claim 1 or 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 in the aqueous medium.

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

5. The method according to any one of the preceding claims, wherein the method further comprises the following steps: - After enzymatic digestion the cells are preferably separated from the aqueous medium by passing the digest through a cell strainer.

6. The method of any of the preceding claims, wherein the matrix metalloproteinase is selected from the group consisting of collagenases, gelatinases, stromelysins, stromelysins, metalloelastases, amelolysins, matrix enzymes, and epidermolysins.

7. A method according to any one of the preceding claims, wherein the matrix metalloprotease is collagenase, preferably wherein the matrix metalloprotease is limited to collagenase, more preferably wherein collagenase is the only protease present in the aqueous medium.

8. A method as claimed in any one of the preceding claims, wherein the aqueous medium comprises dissolved CaCl2.

9. The method of any one of the preceding claims, wherein the tissue sample is a harvested or biopsied tissue sample, optionally a harvested or biopsied tissue sample that is minced prior to the step of contacting the aqueous medium with the tissue sample.

10. The method of any one of claims 3-9, wherein the cells are chondrocytes, with or without their pericellular matrix, and preferably the tissue sample is an articular cartilage tissue sample.

11. An aqueous medium comprising (i) a matrix metalloproteinase and (ii) Ca in a concentration of at least 2 mmol / L. 2+ cation.

12. An aqueous medium as claimed in claim 11, wherein the matrix metalloproteinase is collagenase, preferably wherein the matrix metalloproteinase is limited to collagenase, more preferably wherein collagenase is the only protease present in the aqueous medium.

13. The aqueous medium according to claim 11 or 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 the degradation of extracellular matrix; wherein the extracellular matrix comprises extracellular matrix proteins, preferably collagen.

15. An aqueous medium as claimed in any one of claims 11 to 13 for use as a medicament.

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

17. An aqueous medium for use according to claim 15, wherein the aqueous medium is used in a method of treating a condition associated with a pathological accumulation of an extracellular matrix, preferably a pathological accumulation of an extracellular matrix in or on the skin or in a body orifice or a cavity associated therewith.

18. The aqueous medium for use according to claim 15, wherein the aqueous medium is used in a method for treating wounds, burns, keloid diseases or retained placenta, or for wound debridement.

19. A method for tenderizing a meat product, comprising the steps of: - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca2+ concentration of at least 2 mmol / L 2+ cation; - contacting said aqueous medium with a meat product comprising extracellular matrix proteins under conditions allowing enzymatic digestion of said extracellular matrix proteins.

20. A method for deboning fish products comprising the steps of: - providing an aqueous medium comprising (i) a matrix metalloproteinase and (ii) a Ca2+ concentration of at least 2 mmol / L 2+ cation; - contacting said aqueous medium with a fish product comprising extracellular matrix proteins under conditions allowing enzymatic digestion of said extracellular matrix proteins.

21. A method for producing a peptide or protein hydrolysate, comprising the steps of: - Providing an aqueous medium as described in any one of claims 11 to 13; - contacting said aqueous medium with a composition comprising said protein under conditions allowing enzymatic digestion of the protein.

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

23. The method of claim 21, wherein the protein hydrolysate is a collagen hydrolysate and the protein is collagen.

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