Composition for isolating muscle stem cells from skeletal muscle tissue, comprising enzyme combination and method for isolating muscle stem cells using same

Abstract

The present invention relates to: a composition for isolating muscle stem cells from skeletal muscle tissue, the composition comprising an enzyme combination; and a method for isolating muscle stem cells using same. More specifically, the present invention provides: a composition for isolating muscle stem cells from skeletal muscle tissue in which an optimal protease combination is used; a method for isolating muscle stem cells from skeletal muscle tissue using same; muscle stem cells isolated by said method; cultured meat obtained by culturing the muscle stem cells; and a food composition comprising same.

Description

Composition for isolating muscle stem cells from skeletal muscle tissue containing an enzyme combination and method for isolating muscle stem cells using the same

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0068752, filed May 27, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for isolating muscle stem cells from skeletal muscle tissue comprising a combination of enzymes and a method for isolating muscle stem cells using the same, and more particularly, to a composition for isolating muscle stem cells from skeletal muscle tissue using an optimal combination of proteolytic enzymes, a method for isolating muscle stem cells from skeletal muscle tissue using the same, muscle stem cells isolated from the method, cultured meat obtained by culturing the muscle stem cells, and a food composition comprising the same.

[0003] According to a recent report published by the Food and Agriculture Organization of the United Nations (FAO), the world's population is expected to increase by 0.6% annually from 7.64 billion in July 2018 to 9.2 billion in 2050. In line with this population growth, human meat consumption is also estimated to reach 465 million tons.

[0004] Accordingly, the Food and Agriculture Organization of the United Nations (FAO) has announced that an additional 200 million tons of meat production is needed annually to meet growing demand. Furthermore, recent outbreaks of livestock diseases, such as mad cow disease and foot-and-mouth disease, as well as cases of pathogens contained in livestock intestines not being killed during processing, leading to various illnesses, are causing significant social problems.

[0005] Recently, 'cultured meat' has been attracting attention as a solution to problems such as population growth and the occurrence of diseases in slaughtered livestock.

[0006] Cultured meat refers to edible meat obtained by extracting cells from living animals and multiplying them using cell engineering technology. It can be considered a branch of cellular agriculture that obtains meat without going through the process of raising livestock. Cultured meat is also called alternative meat or artificial meat, and is called in vitro meat because it is grown in a test tube, clean meat because it is produced in a clean production facility rather than a traditional livestock facility, and lab-grown meat because some prototypes are made in a laboratory.

[0007] Cultured meat utilizes tissue engineering, a cell engineering technique. Cells are obtained from living animals, but the process involves painlessly harvesting and culturing stem cells. Therefore, cultured meat represents an ideal food production technology that preserves animal health while also reducing environmental pollution.

[0008] Producing cultured meat requires isolating muscle stem cells from animal skeletal muscle tissue. Skeletal muscle is composed of various cells and the extracellular matrix (ECM), a complex meshwork primarily composed of collagen, laminin, fibronectin, proteoglycans, and elastin. Because the skeletal muscle ECM influences cell survival, proliferation, and differentiation, it is crucial to create an in vivo-like environment when culturing cells in vitro. This has a significant impact on the development of cultured meat.

[0009] Existing muscle stem cell isolation methods involve treating muscle tissue with proteolytic enzymes, which are commonly used for dissociating muscle fibers and single cells. For example, Non-Patent Document 1 discloses a method for dissociating muscle tissue from rat hindlimb muscles by sequentially treating them with collagenase ± and trypsin. However, the method does not disclose the effects of this enzyme combination on cell proliferation, purity, or differentiation.

[0010] Accordingly, the inventors of the present invention have made efforts to isolate high-purity muscle stem cells with maintained functional characteristics of cells by considering the structure of ECM in skeletal muscle tissue, and as a result, have completed the present invention by identifying an optimal enzyme combination capable of isolating muscle stem cells that can be effectively used in cultured meat production by improving cell differentiation ability without affecting cell proliferation and purity.

[0011] [Prior Art Literature]

[0012] [Non-patent literature]

[0013] (Non-patent document 0001) (Non-patent document 1) In Vitro Cell Dev Biol Anim. 2011 Aug;47(7):454-8.

[0014] Accordingly, an object of the present invention is to provide a composition for isolating muscle stem cells from skeletal muscle tissue using a protein enzyme combination.

[0015] Another object of the present invention is to provide a method for isolating muscle stem cells from skeletal muscle tissue using the aforementioned composition.

[0016] Another object of the present invention is to provide muscle stem cells isolated by the above-described method.

[0017] Another object of the present invention is to provide cultured meat obtained by culturing the aforementioned muscle stem cells and a food composition containing the same.

[0018] Another object of the present invention is to provide a method for producing cultured meat using the method of cross-differentiating the aforementioned muscle stem cells into fat cells, and cultured meat produced therefrom.

[0019] Another object of the present invention is to provide a food composition comprising the cultured meat described above.

[0020] To solve the above-described problem, the present invention provides a composition for isolating muscle stem cells from skeletal muscle tissue, comprising collagenase I, II, IV and trypsin.

[0021] In addition, the present invention provides a composition for use in isolating muscle stem cells from skeletal muscle tissue, comprising collagenase I, II, IV and trypsin.

[0022] Additionally, the present invention provides the use of a composition comprising collagenase I, II, IV and trypsin for the preparation of a kit for isolating muscle stem cells from skeletal muscle tissue.

[0023] In the present invention, the collagenase I may be included at a concentration of 0.05% (w / v) to 2% (w / v).

[0024] In the present invention, the collagenase II may be included at a concentration of 0.05% (w / v) to 2% (w / v).

[0025] In the present invention, the collagenase IV may be included at a concentration of 0.05% (w / v) to 2% (w / v).

[0026] In the present invention, the trypsin may be included at a concentration of 0.05% (w / v) to 2% (w / v).

[0027] In the present invention, the concentration ratio of collagenase I: collagenase II: collagenase IV: trypsin may be 1 to 8: 1 to 8: 1 to 8: 1 to 8: 1 to 8.

[0028] The present invention also provides a method for isolating muscle stem cells from skeletal muscle tissue using the composition described above.

[0029] In the present invention, the method may include the following steps (a) and (b):

[0030] (a) treating the composition to skeletal muscle tissue and pulverizing the tissue or treating the composition after pulverizing the skeletal muscle tissue; and

[0031] (b) Step of culturing the crushed tissue.

[0032] In the present invention, the composition treated in step (a) may be further treated in step (b).

[0033] In the present invention, the method can be performed for 30 to 240 minutes.

[0034] In the present invention, the method may additionally include the following steps (c) and (d) after step (b):

[0035] (c) obtaining a cell population containing muscle stem cells after inactivating the enzyme in the cultured tissue; and

[0036] (d) A step of isolating muscle stem cells into single cells from the obtained cell population.

[0037] In addition, the present invention provides muscle stem cells isolated by the method described above.

[0038] In the present invention, the muscle stem cells may be in the form of a single cell or a cell population including muscle stem cells.

[0039] In the present invention, the cell population including the muscle stem cells may exhibit the following characteristics (i) to (v):

[0040] (i) increased cell viability, (ii) increased cell vitality, (iii) increased total cell number, (iv) greater than 99% purity, and (v) increased stemness.

[0041] Furthermore, the present invention provides cultured meat obtained by culturing the aforementioned muscle stem cells and a food composition containing the same.

[0042] The composition for isolating muscle stem cells according to the present invention and the method for isolating muscle stem cells using the same can efficiently isolate muscle stem cells that maintain functional characteristics from skeletal muscle tissue into single cells or a high-purity cell population using an optimal combination of enzymes, and thus can be effectively utilized in cultured meat.

[0043] Figure 1 is a schematic diagram showing the entire process of extracting muscle stem cells of the present invention.

[0044] Figure 2 shows changes in muscle stem cell viability in cell populations obtained by treating skeletal muscle tissue with various enzyme combinations.

[0045] Figure 3 shows changes in muscle stem cell vitality in cell populations obtained by treating skeletal muscle tissue with various enzyme combinations.

[0046] Figure 4 shows the change in total cell number in cell populations obtained by treating skeletal muscle tissue with various enzyme combinations.

[0047] Figure 5 shows the change in the proportion of muscle stem cells (muscle stem cell purity) in cell populations obtained by treating skeletal muscle tissue with various enzyme combinations.

[0048] Figure 6 shows the differences in myofiber differentiation of muscle stem cells in cell populations obtained by treating skeletal muscle tissue with various enzyme combinations.

[0049] Figure 7 shows the results of karyotype analysis performed to confirm whether the isolated muscle stem cells have a normal number of chromosomes.

[0050] Hereinafter, the present invention will be described in more detail.

[0051] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention.

[0052] All numbers expressing the sizes, quantities, and physical properties of features used in this specification and claims are to be understood as being modified by the term "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and claims are approximations that may vary depending on the desired properties sought to be achieved by those skilled in the art utilizing the teachings disclosed herein.

[0053] The present inventors have derived an optimal enzyme combination that does not negatively affect cell proliferation and purity, but significantly improves differentiation potential, by considering the composition of ECM in skeletal muscle tissue, and confirmed that the cell population isolated using this optimal enzyme combination is a highly pure muscle stem cell population with a muscle stem cell ratio of more than 99%, and has a larger cell number, a higher ratio of living cells (cell viability) and healthy cells (cell vitality), and a higher ratio of cells exhibiting stemness compared to the cell population isolated using only protease or a combination of proteases.

[0054] Accordingly, the first aspect of the present invention relates to a composition for isolating muscle stem cells from skeletal muscle tissue comprising an optimal combination of proteolytic enzymes, wherein the optimal combination of proteolytic enzymes may include collagenases I, II, IV and trypsin.

[0055] In relation to the first aspect, the present invention provides a composition for use in isolating muscle stem cells from skeletal muscle tissue, comprising collagenase I, II, IV and trypsin.

[0056] In relation to the first aspect, the present invention further provides the use of a composition comprising collagenase I, II, IV and trypsin for the manufacture of a kit for isolating muscle stem cells from skeletal muscle tissue.

[0057] Although certain aspects are described herein with the term "comprising", it should be understood that other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0058] The term "collagenase" as used herein refers to an enzyme that decomposes collagen, one of the ECMs, and may generally be extracted from Clostridium histolyticum, but is not limited thereto. Preferably, the composition according to the present invention may include a combination of collagenases I, II, and IV.

[0059] When the composition according to the present invention includes a different type of collagenase, the percentage of living cells (viability), the percentage of healthy cells (vitality), the total number of cells, and the percentage of muscle stem cells exhibiting stem cell potential within the cell population isolated from skeletal muscle tissue may be lower than intended.

[0060] The term "trypsin" used in the present invention refers to a protein-decomposing enzyme secreted from the pancreas, which hydrolyzes the terminals of arginine and lysine among amino acid sequences. The trypsin included in the composition of the present invention may be trypsin isolated from the pancreas of an animal such as a human, cow, pig, etc., or may be a recombinant trypsin that is widely used in the art and readily available.

[0061] The composition according to the present invention may be characterized in that it does not contain dispase and / or pronase. When the composition according to the present invention contains a combination of proteolytic enzymes other than collagenase I, II, IV and trypsin, the ratio of living cells (survival rate), the ratio of healthy cells (vitality), the total number of cells and the ratio of muscle stem cells exhibiting stem cell potential in the cell population isolated from skeletal muscle tissue may be lower than intended, and a problem may arise that a higher concentration of the enzyme combination must be used to exhibit the same effect as the composition of the present invention. For example, when dispase is used instead of trypsin in the composition of the present invention or dispase is additionally included in the composition of the present invention, even if the enzyme combination is used at a higher concentration than the enzyme combination of the present invention, the number of cells in the cell population isolated from skeletal muscle tissue, the ratio of healthy cells and the ratio of muscle stem cells exhibiting stem cell potential may be drastically reduced.

[0062] In the present invention, the composition may contain collagenase I at a concentration of 0.05% (w / v) to 2% (w / v), preferably 0.05% (w / v) to 1.5% (w / v).

[0063] In the present invention, the composition may contain collagenase II at a concentration of 0.05% (w / v) to 2% (w / v), preferably at a concentration of 0.05% (w / v) to 1.5% (w / v).

[0064] In the present invention, the composition may contain collagenase IV at a concentration of 0.05% (w / v) to 2% (w / v), preferably 0.05% (w / v) to 1.5% (w / v).

[0065] In the present invention, the composition may contain trypsin at a concentration of 0.05% (w / v) to 2% (w / v), preferably 0.05% (w / v) to 1.5% (w / v).

[0066] In the present invention, the concentration ratio of collagenase I: collagenase II: collagenase IV: trypsin may be 1 to 8: 1 to 8: 1 to 8: 1 to 8: 1 to 8.

[0067] In the present invention, the composition preferably contains the four enzymes in equal concentration ratios. However, any enzyme combination within the above concentration ratio range may be used without limitation. Combinations of enzymes outside the above concentration ratios may result in excessively low or high concentrations of specific enzymes, which may negatively impact the viability, vitality, cell count, and stem cell capacity of isolated muscle stem cells.

[0068] In the present invention, the total concentration of the combination of collagenases I, II, IV and trypsin in the composition may be 0.2% (w / v) to 8% (w / v). If the composition contains the enzyme combination at a concentration of less than 0.2% (w / v), a problem of a low recovery rate of muscle stem cells may occur, and if the composition contains the enzyme combination at a concentration exceeding 8% (w / v), the ratio of living cells in the separated cell population, the ratio of healthy cells, the total number of cells, and the ratio of muscle stem cells exhibiting stem cell potential may decrease.

[0069] In a specific embodiment of the present invention, the cell viability, cell vitality, and cell count of a cell population isolated using various enzymes, either singly or in combination, were evaluated. As a result, as confirmed in Figures 2 to 4, the cell population isolated using the enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA showed the highest percentage of viable and healthy cells, and the total cell count also increased significantly.

[0070] From a comprehensive perspective, when using an enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA, not only can the cell population containing the largest number of muscle stem cells be isolated from skeletal muscle, but the isolated cell population also exhibits excellent cell viability and cell vitality, indicating that collagenase I, collagenase II, collagenase IV, and trypsin are the optimal combination for isolating muscle stem cells most effectively.

[0071] The composition of the present invention may be used in a buffer solution. The basic buffer solution that may be used in the present invention may be, but is not limited to, PBS (phosphate-buffered saline), TBS (Tris-buffered saline), DPBS (Dulbecco's phosphate buffered saline), or HBSS (Hank's balanced salt solution).

[0072] The composition of the present invention can be used as included in “medium”, “culture medium”, “culture medium”, “medium composition”, “culture composition”, and “culture medium composition”.

[0073] In the present invention, the terms "medium", "culture medium", "culture medium", "medium composition", "culture composition", and "culture medium composition" refer to a culture solution containing nutrients that can support the growth and survival of stem cells under in vitro culture conditions, and are not distinguished in the present specification and may be used interchangeably. The basic medium that can be used in the present invention may be selected from DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-modified Minimum Essential Media), GMEM (Glasgow's Minimal essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), etc., but is not limited thereto.

[0074] In addition, the medium composition of the present invention preferably includes antibiotics, antifungals, and / or substances commonly used in the industry to prevent the growth of mycoplasma to prevent infection by bacteria, fungi, etc. As antibiotics, all antibiotics commonly used in cell culture, such as penicillin-streptomycin, can be used, and as antifungals, arforelicin B can be used, and as mycoplasma inhibitors, commonly used substances, such as gentamicin, ciprofloxacin, and azithromycin, can be used, but are not limited thereto. In addition, a commercially available antibiotic-antimycotic (AA) (Gibco) can be used.

[0075] The term "muscle stem cell" as used herein encompasses precursor cells such as quiescent satellite cells and activated satellite cells called myoblasts. Muscle stem cells can differentiate into muscle cells, and livestock-derived muscle stem cells, in particular, can be used in actual cultured meat production, making them highly valuable in agrobiological applications.

[0076] The composition comprising collagenase I, II, IV and trypsin of the present invention can be used for the manufacture of a kit for isolating muscle stem cells from skeletal muscle tissue. In this case, in addition to the composition comprising collagenase I, II, IV and trypsin described above, the kit can include other components or structures for isolating muscle stem cells from skeletal muscle tissue, and optionally can further include one or more of the following:

[0077] (i) auxiliary reagents (e.g., BSA, antioxidants, buffers, etc.) to improve cell viability or increase separation efficiency;

[0078] (ii) enzyme inhibitor or neutralizing solution to stop enzyme activity after tissue decomposition;

[0079] (iii) Antibodies or magnetic beads for recognition of markers of isolated cells (for MACS or FACS);

[0080] (iv) a cell strainer to help separate single cells;

[0081] (v) Buffer solution for cell storage or washing (PBS, HEPES buffer, etc.),

[0082] (vi) staining reagents or assay kits for measuring or quantifying the viability of isolated cells, and

[0083] (vii) Protocol documents, pre-filled tubes or other laboratory equipment to assist in the use of other kits.

[0084] Since muscle stem cells, which are the main raw material of cultured meat, can be efficiently separated from skeletal muscle tissue using the composition according to the present invention, the composition of the present invention can be used in a method of isolating muscle stem cells by treating skeletal muscle tissue under certain conditions.

[0085] Accordingly, the second aspect of the present invention relates to a method for isolating muscle stem cells from skeletal muscle tissue using the composition for isolating muscle stem cells described above, and muscle stem cells isolated using the method.

[0086] In the present invention, the method may include the following steps (a) and (b):

[0087] (a) treating the composition to skeletal muscle tissue and pulverizing the tissue or treating the composition after pulverizing the skeletal muscle tissue; and

[0088] (b) Step of culturing the crushed tissue.

[0089] In the present invention, the composition may be further processed in step (b).

[0090] In the present invention, the method can be performed for 30 to 240 minutes.

[0091] Specifically, the composition may be treated before or after pulverization of skeletal muscle tissue and maintained at a constant concentration for a certain period of time until culture is completed. For example, the time required to complete steps (a) and (b) may be a total of 30 to 240 minutes. Preferably, the treatment may be performed for 30 to 120 minutes, and more preferably, for 60 to 120 minutes. If the composition according to the present invention is treated to skeletal muscle tissue for less than 30 minutes, sufficient dissociation of the muscle tissue may not occur, resulting in a significant decrease in the number of isolated muscle stem cells. If the composition according to the present invention is treated for more than 240 minutes, the isolated muscle stem cells may not exhibit the unique functional characteristics of stem cells due to excessive muscle dissociation.

[0092] Additionally, the composition comprising the enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA may be further processed to maintain a certain concentration before performing culture after tissue grinding. In this case, the composition to be further processed has the same composition as the composition to be processed in step (a), and therefore, its description is omitted.

[0093] In the present invention, the crushing in step (a) can be performed by applying physical force to the tissue before or after the enzyme is treated. At this time, the physical dissociation can be performed by a tool such as a scalpel, scissors, or a homogenizing device, an ultrasonic treatment device, or the like, but is not limited thereto.

[0094] In the present invention, step (b) is a step of culturing skeletal muscle tissue, which has undergone chemical treatment through enzyme treatment and physical treatment through grinding, for a certain period of time to ensure stable enzymatic decomposition. Since the incubation time corresponds to the time of enzyme treatment, it is preferable to incubate for a period of time corresponding to the aforementioned enzyme treatment time.

[0095] In the present invention, the culture in step (b) may be a shaking culture or a static culture, but is not limited thereto.

[0096] According to one embodiment of the present invention, the method may include the following steps (a) to (d) to isolate muscle stem cells as single cells from skeletal muscle tissue:

[0097] (a) a step of treating the composition to skeletal muscle tissue and pulverizing the tissue or treating the composition after pulverizing the skeletal muscle tissue;

[0098] (b) a step of culturing the pulverized tissue;

[0099] (c) obtaining a cell population containing muscle stem cells after inactivating the enzyme in the cultured tissue; and

[0100] (d) A step of isolating muscle stem cells into single cells from the obtained cell population.

[0101] Since the description of steps (a) and (b) above is the same as described above, its description is omitted.

[0102] In the present invention, step (c) may be performed by dissociating muscle stem cells from the tissue and then treating the serum to inactivate the enzyme. If the enzyme activity is not inhibited by the serum treatment, a separate washing step may be performed to remove the enzyme.

[0103] In the present invention, the step (d) is a step performed to separate muscle stem cells into single cells after inactivating the enzyme, and can be separated through various methods, such as a cell separation method using the physical and / or cell physiological characteristics of muscle stem cells or a cell separation method using a marker protein on the cell surface.

[0104] In the present invention, the method for isolating the muscle stem cells includes, but is not limited to, single cell separation through filtering and / or differential centrifugation, density gradient centrifugation, a separation technique based on the difference in cell attachment time (pre-plating technique), cytochalasin B treatment, and a cell sorting method based on antigen-antibody reaction of a marker factor (FACS / MACS system).

[0105] Preferably, the cell population containing muscle stem cells obtained in the step (c) is a high-purity muscle stem cell population having a muscle stem cell ratio of 99% or more, so that individual muscle stem cells can be obtained by performing only single cell separation through filter and / or differential centrifugation without a separate process of purifying only muscle stem cells from the cell composition.

[0106] Muscle stem cells isolated by the method according to the present invention may be in the form of a single cell or a cell population comprising muscle stem cells.

[0107] In the present invention, the cell population including the muscle stem cells exhibits at least one of the following characteristics (i) to (v), and the cell population and single cells isolated therefrom are useful as a main raw material for producing cultured meat:

[0108] (i) increased cell viability, (ii) increased cell vitality, (iii) increased total cell number, (iv) greater than 99% purity, and (v) increased stemness.

[0109] The above cell viability refers to the percentage of living cells within a cell population. The cell population obtained according to the method of the present invention may exhibit increased cell viability compared to a cell population obtained by a single proteolytic enzyme treatment. For example, the cell population may exhibit a cell viability of 95% or greater.

[0110] The above cell vitality refers to the proportion of healthy cells within a cell population and can be determined through a cell vitality assay that measures the level of reduced thiol in cells. The remaining cells, excluding healthy cells within a cell population, can be interpreted as exhibiting apoptosis due to oxidative stress. The cell population obtained according to the method of the present invention can exhibit increased cell vitality compared to a cell population obtained by a single protease treatment. For example, the cell population may have a healthy cell proportion of 95% or more, 96% or more, or 97% or more, which means that the proportion of dead cells within the cell population is less than 5%, less than 4%, or less than 3%.

[0111] The total cell count refers to the total number of cells in a cell population. The cell population obtained according to the method of the present invention may exhibit an increased total cell count compared to a cell population obtained by a single proteolytic enzyme treatment. For example, the cell population may include a cell count increased by 6% to 300% compared to a cell population obtained by a single proteolytic enzyme treatment.

[0112] The purity above refers to the proportion of muscle stem cells within a cell population. For example, the proportion of muscle stem cells within a cell population obtained according to the method of the present invention may be about 99% or greater, preferably 99.5% or greater, 99.6% or greater, or 99.7% or greater.

[0113] The above stem cell potential refers to the differentiation ability of muscle stem cells to differentiate into muscle cells, and increased stem cell potential indicates an increase in the proportion of muscle stem cells with stem cell potential within a cell population. The cell population obtained according to the method of the present invention may have an increased proportion of muscle stem cells with stem cell potential compared to a cell population obtained by a single protease treatment. For example, the cell population may exhibit a 30% to 200% increase in stem cell potential compared to a cell population obtained by a single protease treatment.

[0114] The single proteolytic enzyme used as the above comparison group may be collagenase or trypsin, but may include any proteolytic enzyme capable of decomposing skeletal muscle tissue, such as dispase or pronase, without limitation. The type of collagenase may be, but is not limited to, collagenase I, collagenase II, or collagenase IV.

[0115] In a specific embodiment of the present invention, in order to evaluate the purity of muscle stem cells in a cell population isolated using an enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA, an antibody capable of detecting CD56 and / or CD29, which are surface markers of muscle stem cells, was treated, and then fluorescence-activated cell sorting (FACS) was performed to identify a cell population expressing CD56 and / or CD29. As a result, as shown in Fig. 5, it was confirmed that the proportion of muscle stem cells in the isolated cell population was 99% or more, confirming that a highly pure cell population having a purity of 99% or more of muscle stem cells as single cells was isolated. Therefore, when muscle stem cells are isolated using an enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA, a highly pure muscle stem cell population with a ratio of muscle stem cells of 99% or more as single cells can be obtained without a separate process of purifying only muscle stem cells from the obtained cell population.

[0116] The proportion of muscle stem cells in the cell population isolated using the composition of the present invention may be, for example, about 99% or more, preferably 99.5% or more, 99.6% or more, or 99.7% or more.

[0117] In another specific embodiment of the present invention, in order to confirm the proportion of muscle stem cells with stem cell potential in a cell population isolated using various enzymes alone or in combination, the isolated cell population was differentiated into myofibroblasts, stained with MyHC (myosin heavy chain), and observed under a fluorescence microscope. As a result, as confirmed in Fig. 6, only in the cell population differentiated using the enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA, long and uniformly shaped muscle fibers were formed overall, indicating that the differentiation potential of muscle stem cells was the best.

[0118] In another specific embodiment of the present invention, the karyotype of muscle stem cells isolated using an enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA was analyzed to evaluate chromosomal abnormalities, genetic stability, and cell line identity. As a result, as confirmed in Fig. 7, it was confirmed that the muscle stem cells isolated using an enzyme combination of collagenase I, collagenase II, collagenase IV, and trypsin-EDTA had the same number of chromosomes (30 pairs) as that of a typical cow, and there was no abnormality in the visualized chromosome structure.

[0119] The method of the present invention can isolate a high-purity (99% or more) muscle stem cell population with excellent muscle fiber differentiation ability, and the muscle stem cells isolated by the method of the present invention can be utilized as a food material for producing cultured meat.

[0120] Accordingly, the third aspect of the present invention relates to cultured meat obtained by culturing the aforementioned muscle stem cells and a food composition containing the same.

[0121] In the present invention, the term “cultivation of muscle stem cells” means that muscle stem cells proliferate while maintaining their differentiation potential, which is a stem cell potential.

[0122] In the present invention, the muscle stem cells are non-human animal muscle stem cells, and may be isolated from chicken, turkey, duck, quail, goose, pigeon, pheasant, ostrich, cow, deer, goat, sheep, horse, llama, camel, pig, rabbit, kangaroo, crocodile, turtle, lobster, salmon, tuna or whale, but are not limited thereto.

[0123] In the present invention, the food composition may be manufactured into snacks, dumplings, fried foods, stir-fried foods, soy sauces, seasonings, powder mixes, breads, beverages, processed canned foods, or processed noodles, but is not limited thereto. The form in which the cultured meat is added to a food may be ground into various particle sizes depending on the purpose of use in the food.

[0124] In the present invention, the cultured meat included in the food composition may be additionally treated with a coloring agent. Coloring agents refer to compounds that give color to food, and in order to reproduce the red meat color of beef or pork, artificial coloring agents, natural coloring agents, natural extracts [e.g., beet root extract, pomegranate fruit extract, cherry extract, carrot extract, red cabbage extract, red seaweed extract], modified natural extracts, natural juices (e.g., beet root juice, pomegranate juice, cherry juice, carrot juice, red cabbage juice, red seaweed juice), modified natural juices, FD&C (Food Drug Cosmetics) Red No. 3 (erythrosin), FD&C Green No. 3 (fast green FCF), FD&C Red No. 40 (allura red AC), FD&C Yellow No. 5 (tartazine), FD&C Yellow No. 6 (sunset yellow FCF), FD&C Blue No. 1 (brilliant blue FCF), FD&C Blue No. 2 (indigotine), titanium dioxide, Annatto, anthocyanins, betanins, beta-APE 8 carotenal, beta-carotene, black currant, burnt sugar, canthaxanthin, caramel, carmine / carminic acid, cochineal extract, curcumin, lutein, carotenoids, monascin, paprika, riboflavin, saffron, turmeric, and combinations thereof can be used, but are not particularly limited thereto. Additionally, a coloring agent such as nitrite and ascorbic acid, erythorbic acid, or a salt thereof that promotes the coloring of the nitrite can be further added as a coloring aid.

[0125] In the present invention, the cultured meat included in the food composition may further contain antioxidants, emulsifiers, salts, etc. to stabilize proteins in order to prevent fat oxidation, color change, or fat separation. The antioxidants, emulsifiers, salts, etc. may be used without limitation as long as they are widely used in the art.

[0126] The food composition comprising the cultured meat of the present invention can also be manufactured into a synthetic food product. In addition to the cultured meat, the synthetic food product may further include minerals, vitamins, supplementary vitamins, essential fatty acids, essential amino acids, enzymes, antioxidants, or a combination of two or more thereof.

[0127] Additionally, synthetic foods containing cultured meat may further include flavoring agents, flavor enhancers, sweeteners, coloring agents, colorants, bleaching agents, preservatives, sterilizers, antioxidants, leavening agents, coagulants, or thickeners.

[0128] The form of the above synthetic food may be a paste, puree, soup, pie, powder, granule, chip, tablet, capsule, or spread.

[0129] The cultured meat produced by the cultured meat production method of the present invention can be specifically processed or manufactured in the form of a meat product. The meat product may include, but is not limited to, sterilized meat products, ham, pressed ham, mixed pressed ham, sausage, mixed sausage, dried sausage (dried mixed sausage), semi-dried sausage (semi-dried mixed sausage), heated and frozen sausage, bacon, dried stored meat, seasoned meat, ground processed meat, packaged meat, and other meat products.

[0130] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0131] [Example 1]

[0132] Confirmation of cell viability according to treatment with various enzyme combinations

[0133] 1-1. Enzyme treatment

[0134] After disinfecting the beef rump tissue with 70% ethanol, any tissue suspected of external contamination was removed on a sterile bench. The tissue was transferred to a culture dish without internal contamination, washed three times with PBS, and then, using sterilized scissors and forceps, unnecessary tissues other than muscle, such as membranes and fat, were removed as much as possible. The tissue was transferred to a culture dish without internal contamination, washed three times with PBS, and then, using sterilized scissors and forceps, unnecessary tissues other than muscle, such as membranes and fat, were removed as much as possible. The tissue was divided into six groups of 2 g each and prepared. The enzyme solutions to be treated in each group were prepared as shown in Table 1. The total enzyme concentration in each solution was adjusted to 0.2% (w / v). Enzyme solutions for groups 5 and 6, which contain combinations of two or more enzymes, were prepared by adding equal amounts of individual enzymes. 2 g of tissue was placed in 10 ml of the enzyme combination solution of each group in Table 1, cut into small pieces in the enzyme solution, and the solution containing the cut pieces was uniformly ground with a grinder. 10 ml of the 0.2% (w / v) enzyme combination solution of each group in Table 1 was added to the ground tissue, and shaking incubation was performed at 37.5°C and 180 rpm for 60 minutes to homogenize the tissue. After treating with 0.1% DNase I for 1 minute, the tissue was filtered through a 100 μm or 70 μm strainer to destroy any remaining red blood cells. The tissue was washed with medium supplemented with 10% FBS, diluted with culture medium, plated, and then cell culture was performed.

[0135] Enzymes contained in 10 ml of group enzyme combination solution 1. Collagenase I 2. Collagenase II 3. Collagenase IV 4. Trypsin-EDTA 5. Collagenase I + Collagenase II + Collagenase IV 6. Collagenase I + Collagenase II + Collagenase IV + Trypsin-EDTA

[0136] 1-2. Check cell viability

[0137] Cells attached to the cell culture dish were treated with trypsin / EDTA to disperse all cells. The cell suspension was mixed to obtain a homogeneous mixture. Representative samples from the cell suspension were aspirated into a microfluidic tube using a pipette. One part of acridine orange stain (AO) / 4',6-diamidino-2-phenylindole (DAPI) was added to 19 parts of the cell suspension and mixed using a pipette. Approximately 10 μl of the sample was added to each well of the slide, and the slide was placed in the analyzer and analyzed for cell viability. After analysis, the results of the displayed viability (%) and total cell concentration (cells / ml) were confirmed. As shown in Figure 2, the cell population obtained when treated with the enzyme combination of Group 6 (collagenase I, collagenase II, collagenase IV, and trypsin-EDTA) in Table 1 showed the highest cell viability compared to the other groups in Table 1.

[0138] [Example 2]

[0139] Cell vitality was confirmed by treatment with various enzyme combinations.

[0140] In order to confirm the cell vitality of the cell population obtained after treating the enzyme in the same manner as in Example 1-1, the following method was performed.

[0141] Cells attached to the cell culture dish were treated with trypsin / EDTA to disperse all cells. The cell suspension was mixed to obtain a homogeneous mixture. Representative samples of the cell suspension were pipetted into a microfluidic tube. One part of the staining reagent (VitaBright-48™) was added to each 19 parts of the cell suspension and mixed using a pipette. Approximately 10 μl of the sample was added to each well of the slide, and the slide was placed in the analyzer for analysis of cell viability. The staining reagent (VitaBright-48™) is used to detect changes in the levels of reduced thiol (GSH) in cells undergoing apoptosis or pathological processes. This staining reagent stains cells in an intensity-dependent manner, reflecting their redox state. Therefore, a high fluorescence intensity in a specific cell indicates a high level of reduced thiol. Reduced thiol levels are indicative of overall health and can be used to assess cell viability. The analysis results are displayed as scatter plots and histograms showing the distribution of thiol levels across the cell population. By comparing the intensity of the staining reagent (VitaBright-48™) between enzyme-treated and control cell samples, the proportion of cells with low vitality, such as cells undergoing apoptosis or under stress, was identified. As shown in Figure 3, cell vitality was also confirmed to be the highest in the cell population obtained when treated with the enzyme combination of Group 6 (collagenase I, collagenase II, collagenase IV, and trypsin-EDTA) compared to the other groups.

[0142] [Example 3]

[0143] Cell counting according to various enzyme combination treatments

[0144] In order to confirm the total number of cells in the cell population obtained after treating the enzyme in the same manner as in Example 1-1 above, the following method was performed.

[0145] Cells attached to the cell culture vessel were treated with trypsin / EDTA to disperse all cells. The cell suspension was mixed to obtain a homogeneous mixture. A representative sample of the cell suspension was aspirated into a microfluidic tube using a pipette. One part PI and AO fluorescent reagents were added to each part of the cell suspension and mixed. The stained sample was injected onto a disposable slide and inserted into a cell counting device to count the cells. Multiple images were captured and analyzed for accurate measurements. After analysis, the total cell number (cells / ml) and cell size (μm) were confirmed. As shown in Figure 4, the total cell number of the cell population obtained when treated with the enzyme combination of Group 6 (collagenase I, collagenase II, collagenase IV, and trypsin-EDTA) in Table 1 was significantly higher than that of the other groups in Table 1.

[0146] [Example 4]

[0147] Confirmation of muscle stem cell purity through treatment with various enzyme combinations

[0148] In order to confirm the ratio (purity) of muscle stem cells isolated from the cell population obtained after treating the enzyme in the same manner as in Example 1-1 above, the following method was performed.

[0149] Cells attached to the cell culture dish were treated with trypsin / EDTA to disperse all cells. Cells to be analyzed were collected and washed with PBS to remove any residual medium or serum. The cell concentration was adjusted to the desired concentration in an appropriate buffer. Typically, a cell concentration of 1 to 2 million cells per ml is suitable for flow cytometry. 100 μl of the cell suspension (containing approximately 100,000 to 200,000 cells) was transferred to a FACS (Fluorescence-activated cell sorting) tube, and the appropriate amount of fluorescently labeled anti-CD56 antibody was added to the tube according to the manufacturer's instructions, and mixed thoroughly by gentle pipetting. The cells were incubated with the CD56 antibody for 15 to 30 minutes at room temperature, protected from light. The appropriate amount of fluorescently labeled anti-CD29 antibody was added to the tube according to the manufacturer's instructions, and mixed thoroughly by gentle pipetting. Cells were incubated with CD29 antibody at room temperature, protected from light, for an additional 15–30 minutes. After antibody staining was complete, cells were washed twice with PBS to remove unbound antibody. Cells were centrifuged at 300–400 × g (times gravity) for 5 minutes, and the supernatant was carefully aspirated to avoid aspirating the cell pellet. The stained cells were resuspended in an appropriate volume of buffer (PBS containing 2% FBS) for flow cytometry analysis. The stained cells were transferred to a flow cytometer sample tube, and the flow cytometer was set up according to the instrument manual, adjusting the laser and detector settings for the fluorophore being used (e.g., FITC for CD56 and PE for CD29). The stained cells were analyzed using an appropriate gating strategy to identify and quantify cell populations expressing CD56 and / or CD29, surface markers of muscle stem cells, and to assess purity.As shown in Fig. 5, when the cell population obtained by treating with the enzyme combination of Group 6 (collagenase I, collagenase II, collagenase IV, and trypsin-EDTA) of Table 1 was treated, the muscle stem cell ratio was 99% or higher, confirming that a high-purity muscle stem cell population could be isolated.

[0150] [Example 5]

[0151] Confirmation of stem cell function through treatment with various enzyme combinations

[0152] In order to confirm the proportion of muscle stem cells with stem cell potential in the cell population obtained after treating the enzyme in the same manner as in Example 1-1, the following method was performed.

[0153] Cells were fixed in 4% paraformaldehyde (PFA) for 10–20 minutes at room temperature, and the remaining fixative was removed. The cells were washed with PBS to completely remove any residue. To facilitate antibody penetration into the cells, cells were incubated in a permeabilizing solution (e.g., 0.05% Triton X-100 in PBS) at room temperature to enhance cell permeabilization. Cells were incubated in blocking solution (5% BSA) for 30–60 minutes at room temperature or overnight at 4°C to block nonspecific binding sites. Primary MyHC antibodies were diluted in blocking solution according to the manufacturer's instructions. Cells were incubated with the diluted primary antibodies for 1–2 hours at room temperature or overnight at 4°C, protected from light. Unbound primary antibodies were removed by washing the cells three times for 5 minutes each with washing buffer (e.g., PBS). Fluorescent-conjugated secondary antibodies were diluted in solution according to the manufacturer's instructions, and cells were incubated with the diluted secondary antibodies for 1 hour at room temperature, protected from light. Unbound secondary antibodies were removed by washing cells three times with washing buffer (e.g., PBS) for 5 minutes each. After nuclear staining with DAPI, cells were washed three times with washing buffer (e.g., PBS) for 5 minutes each to remove unbound DAPI solution. The edges of the coverslip were fixed with nail polish or mounting solution. After the mounting solution attached to the coverslip was completely dried, the immunostained cells were visualized using a fluorescence microscope or confocal microscope equipped with an appropriate filter set. As shown in Fig. 6, compared to the other groups in Table 1, only the cell population obtained when treated with the enzyme combination of Group 6 (collagenase I, collagenase II, collagenase IV, and trypsin-EDTA) in Table 1 showed the best myofiber differentiation ability, with long and uniformly shaped myofibers formed overall.

[0154] [Example 6]

[0155] Cell karyotype confirmation

[0156] To evaluate chromosomal aberrations, genetic stability and cell line identity of muscle stem cells isolated using a combination of enzymes from Group 6 (collagenase I, collagenase II, collagenase IV and trypsin-EDTA), cell karyotype analysis was performed as follows.

[0157] When bovine muscle stem cells were approximately 80% distributed in the T-flask, metaphase cells were arrested. The cultured cells were treated with a colcemid solution at a concentration of 0.1 to 0.2 μg / mL and incubated at 37°C for 2 to 4 hours. After confirming that chromosomes were condensed for analysis, the colcemid solution was treated and the cells were detached from the culture dish using trypsin or a similar enzyme. The cells were collected in a centrifuge tube and centrifuged at low speed (e.g., 200 to 300 × g) for 5 minutes to pellet the cells. The cell pellet was resuspended in a hypotonic solution (e.g., 0.075 M KCl) and incubated at room temperature for 10 to 20 minutes to swell the cells and promote chromosome spreading. After hypotonic solution treatment, the cells were fixed by adding a fixative dropwise while gently mixing the suspension. The fixed cells were incubated at room temperature for 10 to 15 minutes or overnight at 4°C. The fixed cell suspension was dropped onto slides and allowed to air dry. The prepared slides were stained using Giemsa stain or other chromosome stains according to the manufacturer's instructions. The slides were rinsed with distilled water and air dried, and then the stained slides were examined under a light microscope equipped with a karyotype analysis system. Chromosome spreads were analyzed to determine the number, size, shape, and banding of chromosomes. Representative metaphase spread images were captured for karyotype analysis and documentation, and chromosome numbers were counted and assessed for abnormalities. In addition, the karyotype was interpreted based on the observed chromosome morphology and number and compared with the expected small karyotype (2n=60). As shown in Figure 7, muscle stem cells isolated using the enzyme combination of Group 6 had a normal chromosome number, including 29 pairs of autosomes (non-sex chromosomes) and one pair of sex chromosomes, and no abnormalities were observed in the visualized chromosome structure.

[0158] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for isolating muscle stem cells from skeletal muscle tissue, comprising collagenases I, II, IV and trypsin.

2. A composition for isolating muscle stem cells from skeletal muscle tissue, wherein the collagenase I is contained in a concentration of 0.05% (w / v) to 2% (w / v) in the first paragraph.

3. A composition for isolating muscle stem cells from skeletal muscle tissue, wherein the collagenase II is contained in a concentration of 0.05% (w / v) to 2% (w / v) in the first paragraph.

4. A composition for isolating muscle stem cells from skeletal muscle tissue, wherein the collagenase IV is contained in a concentration of 0.05% (w / v) to 2% (w / v) in the first paragraph.

5. A composition for isolating muscle stem cells from skeletal muscle tissue, wherein the trypsin is contained at a concentration of 0.05% (w / v) to 2% (w / v) in the first paragraph.

6. A composition for isolating muscle stem cells from skeletal muscle tissue, wherein the concentration ratio of collagenase I: collagenase II: collagenase IV: trypsin in paragraph 1 is 1 to 8: 1 to 8: 1 to 8: 1 to 8: 1 to 8.

7. A method for isolating muscle stem cells from skeletal muscle tissue using the composition of any one of claims 1 to 6.

8. In the 7th paragraph, the method is a method for isolating muscle stem cells from skeletal muscle tissue, comprising the following steps: (a) treating the composition to skeletal muscle tissue and pulverizing the tissue or treating the composition after pulverizing the skeletal muscle tissue; and (b) Step of culturing the crushed tissue.

9. A method for isolating muscle stem cells from skeletal muscle tissue, wherein in step (b), the composition treated in step (a) is further treated.

10. A method for isolating muscle stem cells from skeletal muscle tissue, wherein the method is performed for 30 to 240 minutes in the 8th paragraph.

11. A method for isolating muscle stem cells from skeletal muscle tissue, further comprising the following steps (c) and (d) after step (b) in paragraph 8: (c) obtaining a cell population containing muscle stem cells after inactivating the enzyme in the cultured tissue; and (d) A step of isolating muscle stem cells into single cells from the obtained cell population.

12. Muscle stem cells isolated by any one of the methods of clauses 7 to 11.

13. In the 12th paragraph, the muscle stem cell is in the form of a single cell or a cell population including muscle stem cells.

14. In the 13th paragraph, the cell population including the muscle stem cells exhibits one or more of the following characteristics (i) to (v): (i) increased cell viability; (ii) increased cell vitality; (iii) increased total cell number; (iv) purity of 99% or more; and (v) Increased stemness.

15. Cultured meat obtained by culturing muscle stem cells of Article 12.

16. A food composition comprising the cultured meat of Article 15.

17. A composition according to any one of claims 1 to 6 for use in isolating muscle stem cells from skeletal muscle tissue.

18. Use of the composition of any one of claims 1 to 6 for the manufacture of a kit for isolating muscle stem cells from skeletal muscle tissue.

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