Composition for separating muscle cells and use thereof

Abstract

The present invention relates to a composition for isolating stem cells and the use thereof. A composition comprising papain according to one aspect can effectively isolate stem cells from tissue, and the isolated stem cells can be used to culture cells or produce cultured meat.

Description

Technology Field

[0001] The present invention relates to a composition for isolating stem cells and the use thereof. Background Technology

[0002] The degradation of extracellular matrix proteins (ECM proteins) constituting the tissue is an essential step for isolating stem cells from tissue. Collagen is known to be the primary ECM protein that makes up muscle. Therefore, effectively degrading muscle-constituting ECM proteins, including collagen, is closely linked to increasing the efficiency of muscle stem cell isolation and recovery.

[0003] In the cultured meat production process, since stem cells are a raw material for manufacturing cultured meat, it is essential that the substances used during the preparation of stem cells be harmless to the human body or fall within the scope of food additives. Accordingly, collagenase and pronase, which are generally used in cell culture, are enzymes purified from bacteria (Clostridium histolyticum, Streptomyces griseus), so there is a high possibility that harmful substances may remain.

[0004] Therefore, there is a need to develop alternative enzymes that can be added to food and have functional equivalence to the aforementioned collagenase and pronase. The problem to be solved

[0005] One aspect provides a composition for isolating stem cells, comprising papain.

[0006] Another aspect provides a method for isolating stem cells, comprising the step of treating tissue isolated from an individual other than a human with papain.

[0007] Another aspect provides a separated stem cell using a method for isolating the above-mentioned stem cell.

[0008] Another aspect provides a cell culture method comprising the step of culturing isolated stem cells using a method for isolating the above-mentioned stem cells.

[0009] Another aspect provides a method for producing cultured meat, comprising the step of culturing isolated stem cells using a method for isolating the stem cells.

[0010] Another aspect provides a use for isolating stem cells from a composition containing papain. means of solving the problem

[0011] One aspect provides a composition for isolating stem cells comprising papain.

[0012] The term "Papain" in this specification refers to a proteolytic enzyme extracted from papaya, known to act as a non-specific proteolytic enzyme.

[0013] In this specification, the term "stem cell" refers to a cell having differentiation ability (potency) and self-renewal ability. Stem cells may be classified into pluripotency, multipotency, or unipotency depending on their differentiation ability.

[0014] The terms "separated" or "separated" in this specification mean existing in an environment different from the environment of naturally occurring cells or tissues.

[0015] The above composition may be for isolating stem cells from animal tissue, and the animal may be one other than a human. The animal may include livestock such as chicken, bovine, pocrine, horse, and sheep, and specifically may be a chicken, a bovine, and / or a pig.

[0016] Accordingly, the above tissue may be derived from one or more selected from the group consisting of chickens, cattle, and pigs, and specifically may be derived from cattle and / or pigs.

[0017] The tissue for isolating the above-mentioned stem cells may be a tissue isolated from an individual and may comprise one or more selected from the group consisting of muscle and fat, specifically comprising muscle. Additionally, the above-mentioned muscle tissue may be derived from one or more selected from the group consisting of neck, loin, striploin, tenderloin, round, sirloin, foreleg, brisket, ribs, shank, etc.

[0018] The above stem cells may include one or more selected from the group consisting of muscle stem cells and adipose stem cells, and specifically may include muscle stem cells.

[0019] In this specification, the term "myogenic stem cell" refers to a cell having the characteristics of a myogenic stem cell, including proliferation without transformation, indefinite proliferation, self-renewal ability, and the ability to differentiate into muscle; any cell exhibiting self-renewal ability, indefinite proliferation ability, or muscle differentiation ability may be included without limitation. The term myogenic stem cell may be used interchangeably with myogenic satellite cells (SCs).

[0020] In one embodiment, the composition may contain 1,000 to 200,000 unit / ml of papain. Specifically, the above composition is 1,000 to 200,000 unit / ml, 1,000 to 180,000 unit / ml, 1,000 to 150,000 unit / ml, 1,000 to 120,000 unit / ml, 1,000 to 100,000 unit / ml, 1,000 to 80,000 unit / ml, 1,000 to 50,000 unit / ml, 1,000 to 40,000 unit / ml, 1,000 to 30,000 unit / ml, 1,000 to 22,000 unit / ml, 1,000 to 25,000 unit / ml, 1,000 to 20,000 unit / ml, 1,500 to 200,000 unit / ml, 1,500 to 180,000 unit / ml, 1,500 to 150,000 unit / ml, 1,500 to 120,000 unit / ml, 1,500 to 100,000 unit / ml, 1,500 to 80,000 unit / ml, 1,500 to 50,000 unit / ml, 1,500 to 40,000 unit / ml, 1,500 to 30,000 unit / ml, 1,500 to 25,000 unit / ml, 1,500 to 22,000 unit / ml, 1,500 to 20,000 unit / ml, 2,000 to 200,000 unit / ml, 2,000 to 180,000 unit / ml, 2,000 to 150,000 unit / ml, 2,000 to 120,000 unit / ml, 2,000 to 100,000 unit / ml, 2,000 to 80,000 unit / ml, 2,000 to 50,000 unit / ml, 2,000 to 40,000 unit / ml, 2,000 to 30,000 unit / ml, 2,000 to 25,000 unit / ml, 2,000 to 22,000 unit / ml, 2,000 to 20,000 unit / ml, 5,000 to 200,000 unit / ml, 5,000 to 180,000 unit / ml, 5,000 to 150,000 unit / ml, 5,000 to 120,000 unit / ml, 5,000 to 100,000 unit / ml, 5,000 to 80,000 unit / ml, 5,000 to 50,000 unit / ml, 5,000 to 40,000 unit / ml, 5,000 to 30,000 unit / ml, 5,000 to 25,000 unit / ml, 5,000 to 22,000 unit / ml, 5,000 to 20,000 unit / ml, 10,000 to 200,000 unit / ml, 10,000 to 180,000 unit / ml, 10,000 to 150,000 unit / ml, 10,000 to 120,000 unit / ml, 10,000 to 100,000 unit / ml, 10,000 to 80,000 unit / ml, 10,000 to 50,000 unit / ml, 10,000 to 40,000 unit / ml, 10,000 to 30,000 unit / ml, 10,000 to 25,000 unit / ml, 10,000 to 22,000 unit / ml, 10,000 to 20,000 unit / ml, 15,000 to 200,000 unit / ml, 15,000 to 180,000 unit / ml, 15,000 to 150,000 unit / ml, 15,000 to 120,000 unit / ml, 15,000 to 100,000 unit / ml, 15,000 to 80,000 unit / ml, 15,000 to 50,000 unit / ml, 15,000 to 40,000 unit / ml, 15,000 to 30,000 unit / ml, 15,000 to 25,000 unit / ml, 15,000 to 22,000 unit / ml, 15,000 to 20,000 unit / ml, 18,000 to 200,000 unit / ml, 18,000 to 180,000 unit / ml, 18,It may contain papain at a concentration of 0.00 to 150,000 unit / ml, 18,000 to 120,000 unit / ml, 18,000 to 100,000 unit / ml, 18,000 to 80,000 unit / ml, 18,000 to 50,000 unit / ml, 18,000 to 40,000 unit / ml, 18,000 to 30,000 unit / ml, 18,000 to 25,000 unit / ml, 18,000 to 22,000 unit / ml, or 18,000 to 20,000 unit / ml.

[0021] In one embodiment, the composition may be a liquid composition (solution) in which papain of the above concentration is dissolved or suspended.

[0022] In one embodiment, the composition may contain papain so that papain at the concentration (1,000 to 200,000 unit / ml) can be treated to animal tissue.

[0023] The term “unit (U)” in this specification is a unit representing the activity of an enzyme, defined as the amount of substrate converted per unit time under given conditions. 1 unit is defined as the amount of enzyme required to convert 1 μmol of substrate into product per minute under optimal conditions.

[0024] In one embodiment, in order to effectively isolate stem cells from animal tissue, the isolated animal tissue may be treated with papain at the above concentration.

[0025] In one embodiment, the composition may be for treating animal tissue for 30 to 90 minutes, specifically for 30 to 90 minutes, 30 to 80 minutes, 30 to 75 minutes, 30 to 70 minutes, 30 to 65 minutes, 30 to 60 minutes, 40 to 90 minutes, 40 to 80 minutes, 40 to 75 minutes, 40 to 70 minutes, 40 to 65 minutes, 40 to 60 minutes, 45 to 90 minutes, 45 to 80 minutes, 45 to 75 minutes, 45 to 70 minutes, 45 to 65 minutes, 45 to 60 minutes, 50 to 90 minutes, 50 to 80 minutes, 50 to 75 minutes, It may be for treating animal tissue for 50 to 70 minutes, 50 to 65 minutes, 50 to 60 minutes, 55 to 90 minutes, 55 to 80 minutes, 55 to 75 minutes, 55 to 70 minutes, 55 to 65 minutes, 55 to 60 minutes, 60 to 90 minutes, 60 to 80 minutes, 60 to 75 minutes, 60 to 70 minutes, or 60 to 65 minutes.

[0026] The above composition may be used in vitro. Therefore, the stem cell isolation may refer to isolation under in vitro conditions.

[0027] Stem cells isolated using the above composition may be used to produce cell-cultured meat.

[0028] In this specification, the term "cultured meat" refers to edible meat obtained by collecting animal cells and multiplying them using cell engineering technology; it can be described as a field of cellular agriculture that obtains meat without the process of raising livestock. In Korean, it is referred to as cultured meat, alternative meat, or artificial meat. In English, it is called in vitro meat (meaning it is grown in a test tube), artificial meat (meaning it is synthesized by humans using stem cells rather than being natural), clean meat (meaning it is produced in clean production facilities rather than traditional livestock facilities), or lab-grown meat (meaning it is sometimes produced in a laboratory).

[0029] In one embodiment, the composition is for isolating stem cells from isolated animal tissue, and specifically, may be for isolating muscle stem cells from isolated animal muscle tissue.

[0031] Another aspect provides a method for isolating stem cells, comprising the step of treating tissue isolated from an individual other than a human with papain or a composition containing papain. The same parts as described above apply equally to the method.

[0032] The individuals other than the human mentioned above may be animals, and specifically may include one or more selected from the group consisting of cattle, chickens, and pigs.

[0033] In one embodiment, the papain or the composition containing papain may be a composition in the form of a liquid in which papain is dissolved or suspended.

[0034] In one embodiment, the step of treating the separated tissue in the method with papain or a composition containing papain may include treating with papain at a concentration of 1,000 to 200,000 unit / ml or a composition containing the same. Specifically, the method comprises 1,000 to 200,000 unit / ml, 1,000 to 180,000 unit / ml, 1,000 to 150,000 unit / ml, 1,000 to 120,000 unit / ml, 1,000 to 100,000 unit / ml, 1,000 to 80,000 unit / ml, 1,000 to 50,000 unit / ml, 1,000 to 40,000 unit / ml, 1,000 to 30,000 unit / ml, 1,000 to 22,000 unit / ml, 1,000 to 25,000 unit / ml, 1,000 to 20,000 unit / ml, and 1,500 to 200,000 unit / ml. 1,500 to 180,000 unit / ml, 1,500 to 150,000 unit / ml, 1,500 to 120,000 unit / ml, 1,500 to 100,000 unit / ml, 1,500 to 80,000 unit / ml, 1,500 to 50,000 unit / ml, 1,500 to 40,000 unit / ml, 1,500 to 30,000 unit / ml, 1,500 to 25,000 unit / ml, 1,500 to 22,000 unit / ml, 1,500 to 20,000 unit / ml, 2,000 to 200,000 unit / ml, 2,000 to 180,000 unit / ml, 2,000 to 150,000 unit / ml, 2,000 to 120,000 unit / ml, 2,000 to 100,000 unit / ml, 2,000 to 80,000 unit / ml, 2,000 to 50,000 unit / ml, 2,000 to 40,000 unit / ml, 2,000 to 30,000 unit / ml, 2,000 to 25,000 unit / ml, 2,000 to 22,000 unit / ml, 2,000 to 20,000 unit / ml, 5,000 to 200,000 unit / ml, 5,000 to 180,000 unit / ml, 5,000 to 150,000 unit / ml, 5,000 to 120,000 unit / ml, 5,000 to 100,000 unit / ml, 5,000 to 80,000 unit / ml, 5,000 to 50,000 unit / ml, 5,000 to 40,000 unit / ml, 5,000 to 30,000 unit / ml, 5,000 to 25,000 unit / ml, 5,000 to 22,000 unit / ml, 5,000 to 20,000 unit / ml, 10,000 to 200,000 unit / ml, 10,000 to 180,000 unit / ml, 10,000 to 150,000 unit / ml, 10,000 to 120,000 unit / ml, 10,000 to 100,000 unit / ml, 10,000 to 80,000 unit / ml, 10,000 to 50,000 unit / ml, 10,000 to 40,000 unit / ml, 10,000 to 30,000 unit / ml, 10,000 to 25,000 unit / ml, 10,000 to 22,000 unit / ml, 10,000 to 20,000 unit / ml, 15,000 to 200,000 unit / ml, 15,000 to 180,000 unit / ml, 15,000 to 150,000 unit / ml, 15,000 to 120,000 unit / ml, 15,000 to 100,000 unit / ml, 15,000 to 80,000 unit / ml, 15,000 to 50,000 unit / ml, 15,000 to 40,000 unit / ml, 15,000 to 30,000 unit / ml, 15,000 to 25,000 unit / ml, 15,000 to 22,000 unit / ml, 15,000 to 20,000 unit / ml,Treatment with papain or a composition containing it at a concentration of 18,000 to 200,000 unit / ml, 18,000 to 180,000 unit / ml, 18,000 to 150,000 unit / ml, 18,000 to 120,000 unit / ml, 18,000 to 100,000 unit / ml, 18,000 to 80,000 unit / ml, 18,000 to 50,000 unit / ml, 18,000 to 40,000 unit / ml, 18,000 to 30,000 unit / ml, 18,000 to 25,000 unit / ml, 18,000 to 22,000 unit / ml, or 18,000 to 20,000 unit / ml It is possible.

[0035] In one embodiment, the step of treating the separated tissue with papain or a composition containing papain in the method may be to treat the separated tissue with a sufficient amount of papain or a composition containing papain so that the separated tissue is completely immersed in the papain or the composition containing papain.

[0036] In one embodiment, the step of treating the separated tissue in the method with papain or a composition containing papain may involve treating 1 ml to 50 ml of papain or a composition containing papain (liquid composition) per 1 g of separated tissue, and specifically, 1 ml to 50 ml, 1 ml to 40 ml, 1 ml to 30 ml, 1 ml to 20 ml, 1 ml to 10 ml, 1 ml to 8 ml, 1 ml to 6 ml, 1 ml to 5 ml, 1 ml to 4 ml, 2 ml to 50 ml, 2 ml to 40 ml, 2 ml to 30 ml, 2 ml to 20 ml, 2 ml to 10 ml, 2 ml to 8 ml, 2 ml to 6 ml, 2 ml to 5 ml, 2 ml to 4 ml, 4 ml to 50 ml, 4 ml to 40 ml, 4 ml to 30 ml per 1 g of separated tissue. It may include treating papain or a composition containing papain in ml, 4 ml to 20 ml, 4 ml to 10 ml, 4 ml to 8 ml, 4 ml to 6 ml, or 4 ml to 5 ml.

[0037] In one embodiment, the step of treating the tissue separated in the method with papain or a composition containing papain may be performed for 30 to 90 minutes, specifically 30 to 90 minutes, 30 to 80 minutes, 30 to 75 minutes, 30 to 70 minutes, 30 to 65 minutes, 30 to 60 minutes, 40 to 90 minutes, 40 to 80 minutes, 40 to 75 minutes, 40 to 70 minutes, 40 to 65 minutes, 40 to 60 minutes, 45 to 90 minutes, 45 to 80 minutes, 45 to 75 minutes, 45 to 70 minutes, 45 to 65 minutes, 45 to 60 minutes, 50 to 90 minutes, 50 to 80 minutes, 50 to It may include treating the separated tissue for 75 minutes, 50 minutes to 70 minutes, 50 minutes to 65 minutes, 50 minutes to 60 minutes, 55 minutes to 90 minutes, 55 minutes to 80 minutes, 55 minutes to 75 minutes, 55 minutes to 70 minutes, 55 minutes to 65 minutes, 55 minutes to 60 minutes, 60 minutes to 90 minutes, 60 minutes to 80 minutes, 60 minutes to 75 minutes, 60 minutes to 70 minutes, or 60 minutes to 65 minutes.

[0038] The above stem cells may include muscle stem cells and / or adipose stem cells, and specifically may include muscle stem cells.

[0039] The above method may further include a step of isolating, recovering, and / or purifying stem cells from papain-treated tissue or a sample containing the same. The step of isolating, recovering, and / or purifying the stem cells may use methods known in the art, such as centrifugation and filtration, but is not limited thereto.

[0040] The above method may be performed under in vitro conditions.

[0041] In one embodiment, the method is for isolating stem cells from isolated animal tissue, and specifically, may be for isolating muscle stem cells from isolated animal muscle tissue.

[0043] Another aspect is to provide isolated stem cells using a method for isolating the above-mentioned stem cells. The same details as those described above apply equally to the above-mentioned stem cells.

[0044] The above stem cells may include muscle stem cells and / or adipose stem cells, and specifically may include muscle stem cells.

[0045] The isolated stem cells mentioned above may be capable of subculture.

[0046] In this specification, the term "passage" refers to replacing the culture vessel or dividing the cell group for culture in a method of continuously culturing generations of cells, specifically stem cells, in a healthy state for a long period of time. One replacement of the culture vessel or one division of the cell group for culture is referred to as one passage. In the present invention, the term passage may be used interchangeably with generation.

[0047] The above-mentioned isolated stem cells may be capable of differentiation.

[0048] The term "differentiation" as used herein refers to the phenomenon in which the structure or function of cells becomes specialized during growth through cell division and proliferation; that is, the change in form or function of biological cells, tissues, etc., to perform the tasks assigned to each. The degree of differentiation into a specific cell type may be measured or determined by methods well known in the art. Furthermore, said differentiation may be confirmed by examining cell morphology using an optical microscope or a confocal microscope while measuring cell surface markers (e.g., staining cells with tissue-specific or cell-marker-specific antibodies) and changes in cell morphology (e.g., nuclear-to-cytoplasmic ratio) using techniques such as flow cytometry or immunocytochemistry, or by measuring changes in gene expression using techniques well known in the art such as polymerase chain reaction (PCR) and gene-expression profiling.

[0049] In one embodiment, the isolated stem cells can be proliferated through subculture and / or differentiated through differentiation culture, so cultured meat can be produced using the isolated stem cells.

[0050] In one embodiment, when the isolated stem cell is a muscle stem cell, the muscle stem cell may be capable of differentiating into a myocyte and / or myotube.

[0051] The above stem cells may be for producing cultured meat, and specifically, cultured meat can be produced by culturing the isolated stem cells.

[0053] Another aspect provides a cell culture method comprising the step of culturing isolated stem cells using a method for isolating the stem cells. The same parts as described above apply equally to the method.

[0054] In this specification, the term "cell culture" refers to the process of artificially growing living cells in vitro under controlled conditions. Additionally, it may involve aseptically removing a portion of individual tissue, breaking down intercellular connecting substances with enzymes to release the suspension, and spreading the resulting suspension onto the flat bottom of a culture dish, such as a bottle or Petri dish, to grow and proliferate the cells.

[0055] In one embodiment, the cell culture method may be a method for culturing cells to produce cultured meat, and specifically, may be a method for proliferating and culturing muscle stem cells to produce cultured meat. Accordingly, cultured meat can be produced using cells cultured by the above method, specifically muscle stem cells.

[0056] The above culture form may be a conventional two-dimensional or three-dimensional culture known in the art. However, a three-dimensional culture may be preferred to realize a tissue similar to actual biological tissue through cell-to-cell interaction. Specific examples of three-dimensional culture include 3D porous scaffolds, scaffold-free platforms using the cells themselves or cell sheet technology, methods of placing cells within a microchip, methods using hydrogels, and methods using bioreactors.

[0058] Another aspect provides a method for producing cultured meat, comprising the step of culturing isolated stem cells using a method for isolating the stem cells. The same parts as those described above apply equally to the method.

[0059] The above-mentioned isolated stem cells may be cells isolated from livestock, specifically cells isolated from livestock such as cattle, pigs, or chickens.

[0060] The above cells may be muscle stem cells, specifically muscle stem cells isolated from the muscle tissue of livestock.

[0061] The above-mentioned culturing step may be a method of culturing cells to produce cultured meat, and specifically may include culturing to proliferate stem cells and / or culturing to differentiate stem cells to produce cultured meat.

[0062] In one embodiment, the method may include the step of differentiating the isolated muscle stem cells and / or cultured muscle stem cells into muscle fibers and / or muscle tissue, specifically, the method may include differentiating the cultured cells into myocytes and / or myotubes, and then differentiating them into muscle fibers and / or muscle tissue.

[0063] The above differentiation step may be performed under conditions comprising one or more selected from the group consisting of a scaffold, a differentiation culture medium, and physical stimuli.

[0064] The culture form of the differentiation step described above may be a conventional two-dimensional or three-dimensional culture known in the art. However, three-dimensional culture may be preferred to realize tissues similar to actual biological tissues through cell-to-cell interactions. Specific examples of three-dimensional culture include 3D porous scaffolds, scaffold-free platforms using the cells themselves or cell sheet technology, methods of placing cells within a microchip, methods using hydrogels, and methods using bioreactors.

[0066] Another aspect provides a method for producing cultured meat comprising: 1) treating a tissue isolated from an individual other than a human with papain or a composition containing papain; 2) isolating, recovering, and / or purifying stem cells from the papain-treated tissue or a sample containing the same; and 3) culturing the isolated stem cells. The same parts as described above apply equally to the method.

[0068] Another aspect is to provide a use for isolating stem cells of papain or a composition containing papain. The same parts as described above apply equally to the said use. Effects of the invention

[0069] A composition containing papain according to one aspect can effectively isolate stem cells from tissue, and the isolated stem cells can be used to culture cells or produce cultured meat. Brief explanation of the drawing

[0070] Figure 1 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to the papain treatment time. Figure 2 is a diagram showing the results of confirming the adhesion ability of cells separated from muscle tissue according to papain treatment time. Figure 3 is a diagram showing the results of confirming the number of muscle stem cells isolated from muscle tissue according to the papain treatment time. Figure 4 is a diagram showing the results of confirming the cumulative growth number of muscle stem cells isolated from muscle tissue according to papain treatment time. Figure 5 is a diagram showing the results of confirming the differentiation ability of muscle stem cells isolated from muscle tissue according to papain treatment time. Figure 6 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to papain treatment concentrations (200, 1000, 2000, or 5000 unit / ml). Figure 7 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to papain treatment concentrations (2000, 3000, 4000, or 5000 unit / ml). Figure 8 is a diagram showing the results of confirming the number of muscle stem cells isolated from muscle tissue according to papain treatment concentration (4000 or 5000 unit / ml). Figure 9 is a diagram showing the results of confirming the differentiation ability of muscle stem cells isolated from muscle tissue according to papain treatment concentration (4000 or 5000 unit / ml). Figure 10 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to papain treatment concentration (2000 or 20000 unit / ml). Figure 11 is a diagram showing the results of confirming the number of muscle stem cells isolated from muscle tissue according to papain treatment concentration (20,000 unit / ml). Figure 12 is a diagram showing the results of confirming the differentiation ability of muscle stem cells isolated from muscle tissue according to papain treatment concentration (20,000 unit / ml). Figure 13 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to papain treatment concentration (20,000 or 200,000 unit / ml). Figure 14 is a diagram showing the results of confirming the number of muscle stem cells isolated from muscle tissue according to papain treatment concentration (20,000 or 200,000 unit / ml). Figure 15 is a diagram showing the results of confirming the differentiation ability of muscle stem cells isolated from muscle tissue according to papain treatment concentration (20,000 or 200,000 unit / ml). Figure 16 is a diagram showing the results of confirming the PAX7 expression level of muscle stem cells isolated from papain-treated muscle tissue. Figure 17 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to bromelain treatment concentrations (2500, 5000, or 20000 unit / ml). Figure 18 is a diagram showing the results of confirming the number of muscle stem cells isolated from muscle tissue according to bromelain treatment concentrations (2500, 5000, or 20000 unit / ml). Figure 19 is a diagram showing the results of observing muscle stem cells isolated from papain-treated pig muscle tissue. Figure 20 is a diagram showing the results of observing adipose stem cells isolated from papain-treated porcine adipose tissue. Specific details for implementing the invention

[0071] The following examples and experimental examples will be explained in more detail. However, these examples and experimental examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples and experimental examples.

[0073] Example 1: Method for isolating bovine or porcine muscle stem cells

[0074] To isolate muscle stem cells from bovine muscle tissue, the following experiment was performed.

[0075] First, bovine muscle tissue was washed with a solution of 1% (v / v) AA (antibiotic-antimycotic) added to DPBS (Dulbecco's phosphate-buffered saline). The washed tissue was cut into small pieces and washed again with a solution containing antibiotics. Subsequently, the tissue was lysed by treating it with an enzyme (bromelain, papain, or pronase) at 37°C for a certain period of time. The tissue lysed by the enzyme was suspended in HG-DMEM (Dulbecco's Modified Eagle Medium, high glucose), washed, and the cell pellet was collected by centrifugation. Afterward, the cells from which red blood cells had been removed were treated with an erythrocyte lytic agent at room temperature for 10 minutes, and then filtered sequentially using 100-, 70-, and 40-μm cell strainers. After centrifuging the filtered cells, the supernatant was centrifuged again to collect the cell pellet, and cells derived from bovine muscle tissue were obtained.

[0076] Next, to recover muscle stem cells from the cells derived from the bovine muscle tissue, the muscle tissue-derived cells were cultured in HG-DMEM medium (bovine muscle stem cell proliferation medium; bMuSC-PM) supplemented with 10% (v / v) FBS (fetal bovine serum), 10 ng / mL rbFGF2 (recombinant bovine FGF2), and 1% (v / v) AA. After 2 hours, the suspension cells, which were muscle stem cells, were transferred to a new flask and cultured for 72 hours in a humidity-controlled environment containing 5% CO2 at 37°C. Subsequently, the medium was replaced with fresh medium every two days and cultured until the cell density reached 70-80%. The cultured cells were recovered using 0.25% trypsin-EDTA and used for subsequent experiments.

[0077] Porcine muscle stem cells were isolated using the same method as bovine muscle stem cells, and porcine muscle stem cell proliferation culture medium (pMuSC-PM) was prepared using a medium composed of Ham's F10, 15% FBS, 1% AA, and 10 ng / mL rbFGF2.

[0079] Example 2: Method for isolating bovine or porcine adipose stem cells

[0080] To isolate stromal vascular fraction (SVF), which are adipose stem cells, from bovine adipose tissue, the following experiment was performed.

[0081] First, bovine white adipose tissue was washed with a solution of DPBS supplemented with 1% (v / v) antibiotic-antimycotic and 10 mg / mL gentamicin. The washed tissue was cut into small pieces and then degraded by treatment with an enzyme (collagenase or papain) at 37°C for 60 minutes. The enzyme-degraded tissue was suspended in a medium (bovine SVF proliferation culture medium, bSVF-PM) supplemented with 10% FBS, 1% antibiotic-antimycotic, and 10 mg / mL gentamicin in HG-DMEM, washed, and then filtered using a 100-μm cell strainer. After centrifugation, the cells were treated with an erythrocyte lytic agent at room temperature for 1 minute, and the cells from which red blood cells had been removed were filtered using a 40-μm cell strainer. Subsequently, the cell pellet was recovered by centrifugation again, resuspended in bovine SVF proliferation medium, and cultured in flasks coated with 0.1% (w / v) fish gelatin. Culture was carried out in a humidity-controlled environment of 37°C and 5% CO2, and the medium was replaced with fresh medium after 1 hour. Thereafter, the culture medium was replaced daily until the cell density reached 70-80%, and the cultured cells were treated with 0.25% trypsin-EDTA to harvest and use for subsequent experiments. Porcine SVF was isolated using the same method as bovine SVF, and the porcine SVF proliferation medium (pSVF-PM) used was a medium composed of DMEM / F12, 10% FBS, 1% antibiotic-antimycotic, and 10 mg / mL gentamicin.

[0083] Example 3: Cell culture method

[0084] Bovine and porcine muscle stem cells were cultured in T75 and T175 flasks, and cell harvesting and culture were performed using DPBS, 0.25% trypsin-EDTA, and either bovine muscle stem cell proliferation medium or porcine muscle stem cell proliferation medium. Cell proliferation was evaluated based on the population doubling (PD) value and the cumulative number of growing cells in each passage; the cumulative number of growing cells was calculated by multiplying the initial cell inoculation number by the population doubling level (PDL) value. To analyze cell purity and differentiation ability, bovine muscle stem cells were prepared by inoculating 1 x 10^5 cells into 24-well plates and 3 x 10^5 cells into 6-well plates, respectively. After 2 days, cell purity was confirmed using the prepared samples, and cell differentiation was induced by replacing the culture medium (HG-DMEM, 1% penicillin-streptomycin, 2.5 mM insulin) and culturing for 4 days.

[0085] Bovine / porous SVFs were also cultured in T75 and T175 flasks, and the cells were cultured using DPBS, 0.25% trypsin-EDTA, and bSVF-PM or pSVF-PM.

[0087] Experimental Example 1: Determination of Optimal Papain Treatment Time for Muscle Stem Cell Isolation

[0088] To isolate muscle stem cells from bovine muscle tissue, papain was intended to be used as an enzyme, and the following experiment was performed to optimize the papain treatment time.

[0089] Specifically, muscle stem cells were isolated from bovine muscle tissue based on the method described in Example 1. Papain at a concentration of 2000 unit / ml was used as the enzyme, and after treatment times of 30, 60, or 90 minutes, the total number of isolated cells, adhesion ability, number of sorted cells (muscle stem cells), number of cell proliferations, and differentiation ability were confirmed. Meanwhile, the control group was treated with pronase, a commonly used enzyme, at a concentration of 2000 unit / ml for 60 minutes.

[0090] As a result of the above experiment, the number of cells isolated immediately after enzyme treatment was low when treated for 30 minutes, but showed a level similar to the control group when treated for 60 or 90 minutes (Fig. 1), and it was confirmed that the cell adhesion ability also showed a level similar to the control group (Fig. 2).

[0091] Next, the number of sorted muscle stem cells isolated according to enzyme treatment was checked. It was found that when papain was treated for 30 minutes, the muscle stem cells did not proliferate and could not be measured, while when papain was treated for 60 minutes, significantly superior muscle stem cell isolation efficiency was observed compared to the 90-minute treatment or the control group (Fig. 3). In addition, as a result of continuing to culture the isolated muscle stem cells, it was confirmed that when papain was treated for 60 minutes, the cumulative number of growing cells was significantly superior compared to other experimental groups and the control group (Fig. 4).

[0092] Next, the differentiation ability of muscle stem cells isolated by the above method was confirmed, and it was confirmed that muscle differentiation proceeded in all experimental and control groups, indicating that treatment with papain as an enzyme does not affect the intrinsic characteristics of the cells (Fig. 5).

[0093] Based on the above results, it can be seen that treating with papain as an enzyme for 60 minutes to isolate muscle stem cells from muscle tissue exhibits significantly superior isolation efficiency.

[0095] Experimental Example 2: Confirmation of optimal papain treatment concentration for muscle stem cell isolation (1)

[0096] To determine the optimal treatment concentration for isolating muscle stem cells from bovine muscle tissue using papain, the following experiment was performed.

[0097] Specifically, based on the method described in Example 1, muscle stem cells were isolated from bovine muscle tissue, and papain was treated as an enzyme for 60 minutes. After treatment at various concentrations, the total number of isolated cells, the number of sorted cells (muscle stem cells), and differentiation ability were confirmed.

[0098] First, the number of isolated cells was checked immediately after enzyme treatment by setting the papain concentration to 200, 1000, 2000, or 5000 unit / mL. It was found that when treated at a concentration of 200 or 1000 unit / mL, the number of isolated cells was low, while when treated at a concentration of 2000 or 5000 unit / mL, the number of isolated cells increased significantly (Fig. 6). In addition, it was confirmed that when treated at papain concentrations of 2000, 3000, 4000, or 5000 unit / mL, a similar level of isolated cell number was observed (Fig. 7).

[0099] Next, the number of sorted muscle stem cells isolated by treating with papain at a concentration of 4000 or 5000 unit / mL was checked, and it was confirmed that the isolation efficiency of muscle stem cells was significantly superior to that of the 2000 unit / mL case shown in Fig. 3 (Fig. 8). It was also confirmed that muscle differentiation proceeded in the muscle stem cells isolated under the above conditions, indicating that treatment with papain at the above concentrations does not affect the intrinsic characteristics of the cells (Fig. 9).

[0100] Based on the above results, it can be seen that muscle stem cells can be effectively isolated from muscle tissue when the papain treatment concentration is in the range of 2000 to 5000 unit / mL.

[0102] Experimental Example 3: Confirmation of optimal papain treatment concentration for muscle stem cell isolation (2)

[0103] To further determine the optimal treatment concentration for isolating muscle stem cells from bovine muscle tissue using papain, the following experiment was performed.

[0104] Specifically, based on the method described in Example 1, muscle stem cells were isolated from bovine muscle tissue, and papain was used as an enzyme for 60 minutes. After treatment at a concentration of 2000 or 20000 unit / mL, the total number of isolated cells, the number of sorted cells (muscle stem cells), and differentiation ability were confirmed.

[0105] First, the number of isolated cells was checked immediately after enzyme treatment, and it was confirmed that when treated at a concentration of 20,000 unit / mL, the number of cells was similar to that when treated at a concentration of 2,000 unit / mL (Fig. 10).

[0106] In addition, when the number of isolated muscle stem cells (sorted cells) was checked, it was confirmed that the isolation efficiency of muscle stem cells was significantly superior to that of the case treated with 2000 unit / mL in Fig. 3 (Fig. 11), and it was confirmed that muscle differentiation proceeded in the muscle stem cells isolated under the above conditions, indicating that treatment with papain at the above concentration does not affect the intrinsic characteristics of the cells (Fig. 12).

[0107] Based on the above results, it can be seen that muscle stem cells can be effectively isolated from muscle tissue even when the papain treatment concentration is 20,000 unit / mL.

[0109] Experimental Example 4: Confirmation of optimal papain treatment concentration for muscle stem cell isolation (3)

[0110] To further determine the optimal treatment concentration for isolating muscle stem cells from bovine muscle tissue using papain, the following experiment was performed.

[0111] Specifically, based on the method described in Example 1, muscle stem cells were isolated from bovine muscle tissue, and papain was used as an enzyme for 60 minutes. After treatment at a concentration of 20,000 or 200,000 unit / mL, the total number of isolated cells, the number of sorted cells (muscle stem cells), and differentiation ability were confirmed.

[0112] First, when checking the number of isolated cells immediately after enzyme treatment, it was confirmed that the number of cells was significantly lower when treated at a concentration of 200,000 unit / mL compared to when treated at a concentration of 20,000 unit / mL, indicating that the cell isolation efficiency was significantly reduced (Fig. 13).

[0113] In addition, when the number of isolated muscle stem cells (sorted cells) was checked, it was confirmed that when treated at a concentration of 200,000 unit / mL, the level of muscle stem cells was significantly lower compared to when treated at a concentration of 20,000 unit / mL (Fig. 14), and it was confirmed that the differentiation ability of muscle stem cells isolated by treatment at a concentration of 200,000 unit / mL was also reduced (Fig. 15).

[0114] Based on the above results, it can be seen that when the papain treatment concentration is 200,000 unit / mL, the efficiency of isolating muscle stem cells from muscle tissue is significantly reduced.

[0116] Experimental Example 5: Confirmation of muscle stem cells isolated using papain

[0117] As confirmed through the above experimental example, the following experiment was performed to verify whether muscle stem cells were effectively isolated from bovine muscle tissue using papain.

[0118] Specifically, muscle stem cells were isolated from bovine muscle tissue based on the method described in Example 1, and 20,000 unit / mL of papain was treated as an enzyme for 60 minutes, and the expression of PAX7 (Paired box protein 7), a marker of muscle stem cells, was confirmed using a fluorescence microscope.

[0119] As a result of the above, it was confirmed that cells isolated from muscle tissue using papain express the PAX7 protein (Fig. 16), and it can be seen that the isolated cells are muscle stem cells.

[0121] Experimental Example 6: Confirmation of Bromelain's Muscle Stem Cell Isolation Efficiency

[0122] To determine whether muscle stem cells can be effectively isolated from muscle tissue using bromelain, an enzyme other than papain, the following experiment was performed.

[0123] Specifically, muscle stem cells were isolated from bovine muscle tissue based on the method described in Example 1. Bromelain was used as an enzyme for 60 minutes, and after treatment at concentrations of 2500, 5000, or 20000 unit / mL, the total number of isolated cells and sorted cells (muscle stem cells) isolated from the tissue were determined. Meanwhile, the control group was treated with pronase, a commonly used enzyme, at a concentration of 2000 unit / mL for 60 minutes.

[0124] As a result of the above experiment, it was confirmed that the number of isolated cells was significantly lower when treated with bromelain compared to when treated with pronase (Fig. 17), and the muscle stem cell isolation efficiency was also significantly lower compared to the control group (Fig. 18).

[0125] Based on the above results, it can be seen that bromelain exhibits significantly low efficiency as an enzyme for isolating muscle stem cells from muscle tissue.

[0127] Experimental Example 7: Evaluation of Papain-Induced Isolation of Porcine Muscle Stem Cells

[0128] To determine whether muscle stem cells could be isolated from pig muscle tissue using papain, the following experiment was performed.

[0129] Specifically, based on the method described in Example 1, muscle stem cells were isolated from porcine muscle tissue, and 20,000 unit / mL of papain was treated as an enzyme for 60 minutes, and the cells isolated from the tissue were observed under a microscope.

[0130] As a result of the above experiment, it was confirmed that muscle stem cells can be effectively isolated from pig muscle tissue in the same way as from bovine muscle tissue (Fig. 19). Based on the above results, it can be seen that muscle stem cells can be isolated from pig muscle tissue using papain.

[0132] Experimental Example 8: Evaluation of Papain-Induced Isolation of Adipose Stem Cells

[0133] To determine whether the stromal vascular fraction (SVF), which is an adipose stem cell, can be isolated from adipose tissue using papain, the following experiment was performed.

[0134] Specifically, based on the method described in Example 1, SVF was isolated from porcine adipose tissue, and papain at concentrations of 2,500, 5,000, or 10,000 unit / mL was treated for 60 minutes. After enzyme treatment, the residual tissue and cells isolated from the tissue were observed. Meanwhile, the control group was treated with collagenase 1, a commonly used enzyme, at a concentration of 0.1% for 60 minutes.

[0135] As a result of the above experiment, it was confirmed that adipose stem cells could be isolated from adipose tissue, but the isolation efficiency was lower than that of the control group, collagenase (Fig. 20).

[0137] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A composition for isolating stem cells comprising papain, wherein the composition is for isolating stem cells from animal tissue, and the tissue is derived from muscle. Claim 2 delete Claim 3 A composition according to claim 1, wherein the tissue is derived from one or more selected from the group consisting of chicken, bovine, and pocrine. Claim 4 delete Claim 5 A composition according to claim 1, wherein the stem cells comprise muscle stem cells. Claim 6 A composition according to claim 1, wherein the composition comprises 1,000 unit / ml to 200,000 unit / ml of papain. Claim 7 A method for isolating stem cells comprising the step of treating a tissue isolated from an individual other than a human with papain, wherein the isolated tissue is derived from muscle. Claim 8 The method of claim 7, wherein the step of treating the separated tissue with papain is performed for 30 to 90 minutes. Claim 9 The method of claim 7, wherein the step of treating the separated tissue with papain is to treat papain at a concentration of 2,000 unit / ml to 200,000 unit / ml or a composition comprising papain at said concentration. Claim 10 A cell culture method comprising the step of culturing isolated stem cells using the method of any one of claims 7 to 9. Claim 11 A method for producing cultured meat comprising the step of culturing isolated stem cells using the method of any one of claims 7 to 9.

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