Composition for isolating stem cells and use thereof

Abstract

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

Description

Composition for isolating stem cells and its use

[0001] The present invention relates to a composition for isolating stem cells and its use.

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

[0003] In the cultured meat production process, stem cells are considered raw materials for cultured meat production. Therefore, it is essential that any substances used in the stem cell preparation process be considered harmless to the human body or fall within the scope of food additives. Collagenase and pronase, commonly used in cell culture, are purified enzymes from bacteria (Clostridium histolyticum and Streptomyces griseus), and therefore have a high risk of residual harmful substances.

[0004] Therefore, there is a need to develop a food additive-compatible substitute enzyme that has functional equivalence to the above-mentioned enzymes such as collagenase and protease.

[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 a non-human organism with papain.

[0007] Another aspect provides isolated stem cells using a method for isolating said stem cells.

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

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

[0010] Another aspect provides a use of a composition comprising papain for isolating stem cells.

[0011] One aspect is to provide a composition for isolating stem cells, comprising papain.

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

[0013] The term "stem cell" in this specification refers to a cell with differentiation potency and self-renewal ability. Depending on differentiation ability, stem cells can be classified as pluripotency, multipotency, or unipotency.

[0014] The term "isolated" or "separated" in this specification means existing in an environment different from the environment of the cell or tissue in which it naturally occurs.

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

[0016] Accordingly, the tissue may be derived from one or more selected from the group consisting of chicken, cow and pig, and may be specifically derived from cow and / or pig.

[0017] The tissue for isolating the above stem cells may be a tissue isolated from an individual, and may include at least one selected from the group consisting of muscle and fat, and may specifically include muscle. In addition, the muscle tissue may be derived from at least one selected from the group consisting of shoulder, sirloin, rib eye, tenderloin, rump, rump, foreleg, brisket, ribs, and shank.

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

[0019] The term "myogenic stem cell" as used herein refers to a cell having the characteristics of a muscle stem cell, including proliferation without transformation, infinite proliferation, self-renewal ability, and the ability to differentiate into muscle, and may include, without limitation, any cell that exhibits self-renewal ability or infinite proliferation ability and muscle differentiation ability. The muscle stem cell may be used interchangeably with muscle satellite cells (SCs).

[0020] In one embodiment, the composition may comprise 1,000 to 200,000 units / ml of papain. Specifically, the composition has an amount of 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 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.

[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 include papain such that the animal tissue can be treated with papain at the concentration (1,000 to 200,000 units / ml).

[0023] The term "unit (U)" in this specification represents the activity of an enzyme, and is defined as the amount of substrate converted per unit time under given conditions. One 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, 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 treating animal tissue 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. Accordingly, 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] The term "cultured meat" in this specification refers to edible meat obtained by extracting animal cells and multiplying them using cell engineering technology, and can be said to be a field of cellular agriculture that obtains meat without going through the process of raising livestock. In Korean, it is called cultured meat, alternative meat, or artificial meat, and in English, it is called in vitro meat meaning that it is grown in a test tube, artificial meat meaning that it is synthesized using stem cells by humans rather than natural ones, clean meat meaning that it is produced in a clean production facility rather than a traditional breeding facility, and lab-grown meat because it is sometimes made in a laboratory.

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

[0030]

[0031] Another aspect provides a method for isolating stem cells, comprising treating tissue isolated from a non-human organism with papain or a composition containing papain. The same portions described above also apply to the method.

[0032] The above non-human entity may be an animal, and may specifically include one or more selected from the group consisting of cows, chickens, and pigs.

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

[0034] In one embodiment, the step of treating the tissue separated from the method with papain or a composition comprising papain may include treating the tissue with papain or a composition comprising the same at a concentration of 1,000 to 200,000 units / ml. Specifically, the method comprises the steps of: 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,Papain or a composition comprising the same 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 This may include processing:

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

[0036] In one embodiment, the step of treating the separated tissue with papain or a composition containing papain in the method may be treating 1 ml to 50 ml of papain or a composition containing papain (liquid composition) per 1 g of the separated tissue, 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 It may comprise treating with 30 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 of papain or a composition comprising papain.

[0037] In one embodiment, the step of treating the tissue separated from 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, It may include treating the isolated tissue for 50 to 75 minutes, 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.

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

[0039] The 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 utilize methods known in the art, such as, but not limited to, centrifugation and filtration.

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

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

[0042]

[0043] Another aspect is to provide isolated stem cells using a method for isolating said stem cells. The same aspects described above also apply to said 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 above separated stem cells may be capable of subculture.

[0046] As used herein, the term "passage" refers to the process of continuously culturing cells, specifically stem cells, for the purpose of maintaining them in a healthy, long-term state, by replacing the culture vessel or dividing a cell population. A single replacement of the culture vessel or dividing a cell population is referred to as one passage. In the present invention, "passage" may be used interchangeably with "generation."

[0047] The above separated stem cells may be capable of differentiation.

[0048] The term "differentiation" as used herein refers to a phenomenon in which cells become specialized in structure or function during their growth through division and proliferation, i.e., cells, tissues, etc. of a living organism change in form or function to perform their respective tasks. Measuring or determining the degree of differentiation into a specific cell type can be performed by methods well known in the art. In addition, the differentiation can be confirmed by examining cell morphology using a light microscope or confocal microscope while measuring changes in cell surface markers (e.g., staining cells with tissue-specific or cell-marker-specific antibodies) and cell morphology (e.g., nuclear / 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 are capable of proliferation through subculture and / or differentiation through differentiation culture, and thus cultured meat can be produced using the isolated stem cells.

[0050] In one embodiment, when the isolated stem cells are muscle stem cells, the muscle stem cells may be capable of differentiating into myocytes and / or myotubes.

[0051] The above stem cells may be used to produce cultured meat, and specifically, the isolated stem cells may be cultured to produce cultured meat.

[0052]

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

[0054] As used herein, the term "cell culture" refers to the process of artificially growing living cells in vitro under controlled conditions. It can also involve aseptically removing a portion of a tissue from an individual, enzymatically degrading intercellular connective tissue, and spreading the resulting suspension onto the flat bottom of a culture dish, such as a bottle or petri dish, to allow the cells to grow and proliferate.

[0055] In one embodiment, the cell culture method may be a method for culturing cells for producing cultured meat, and specifically, may be a method for proliferating and culturing muscle stem cells for producing cultured meat. Accordingly, cultured meat can be produced using cells cultured using the 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, three-dimensional culture may be preferable to realize tissues similar to actual living tissues through cell-to-cell interactions. Specific examples of three-dimensional culture include 3D porous scaffolds, scaffold-free platforms using cells themselves or cell sheet technology, methods for arranging cells within microchips, methods using hydrogels, and methods using bioreactors.

[0057]

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

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

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

[0061] The above culturing step may be a method of culturing cells for producing cultured meat, and may specifically include culturing for proliferating stem cells and / or culturing for differentiating stem cells for producing cultured meat.

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

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

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

[0065]

[0066] Another aspect provides a method for producing cultured meat, comprising: 1) treating tissue isolated from a non-human organism 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 also apply to the method.

[0067]

[0068] Another aspect provides a use for isolating stem cells using papain or a composition containing papain. The same principles described above apply to this use.

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

[0070] Figure 1 is a diagram showing the results of confirming the number of cells separated from muscle tissue according to the papain treatment time.

[0071] Figure 2 is a diagram showing the results of confirming the adhesive ability of cells separated from muscle tissue according to the papain treatment time.

[0072] 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.

[0073] Figure 4 is a diagram showing the results of confirming the cumulative growth cell number of muscle stem cells isolated from muscle tissue according to the papain treatment time.

[0074] Figure 5 is a diagram showing the results of confirming the differentiation potential of muscle stem cells isolated from muscle tissue according to the papain treatment time.

[0075] Figure 6 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to the papain treatment concentration (200, 1000, 2000, or 5000 unit / ml).

[0076] Figure 7 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to the papain treatment concentration (2000, 3000, 4000, or 5000 unit / ml).

[0077] Figure 8 is a diagram showing the results of confirming the cell number of muscle stem cells isolated from muscle tissue according to the papain treatment concentration (4000 or 5000 unit / ml).

[0078] Figure 9 is a diagram showing the results of confirming the differentiation ability of muscle stem cells isolated from muscle tissue according to the papain treatment concentration (4000 or 5000 unit / ml).

[0079] Figure 10 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to the papain treatment concentration (2000 or 20000 unit / ml).

[0080] Figure 11 is a diagram showing the results of confirming the cell number of muscle stem cells isolated from muscle tissue according to the papain treatment concentration (20,000 units / ml).

[0081] Figure 12 is a diagram showing the results of confirming the differentiation ability of muscle stem cells isolated from muscle tissue according to the papain treatment concentration (20,000 units / ml).

[0082] Figure 13 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to the papain treatment concentration (20,000 or 200,000 unit / ml).

[0083] Figure 14 is a diagram showing the results of confirming the cell number of muscle stem cells isolated from muscle tissue according to the papain treatment concentration (20,000 or 200,000 unit / ml).

[0084] Figure 15 is a diagram showing the results of confirming the differentiation ability of muscle stem cells isolated from muscle tissue according to the papain treatment concentration (20,000 or 200,000 unit / ml).

[0085] Figure 16 is a diagram showing the results of confirming the PAX7 expression level of muscle stem cells isolated from muscle tissue treated with papain.

[0086] Figure 17 is a diagram showing the results of confirming the number of cells isolated from muscle tissue according to the bromelain treatment concentration (2500, 5000, or 20000 unit / ml).

[0087] Figure 18 is a diagram showing the results of confirming the cell number of muscle stem cells isolated from muscle tissue according to the bromelain treatment concentration (2500, 5000, or 20000 unit / ml).

[0088] Figure 19 is a drawing showing the results of observing muscle stem cells isolated from pig muscle tissue treated with papain.

[0089] Figure 20 is a drawing showing the results of observing adipose stem cells isolated from porcine adipose tissue treated with papain.

[0090] The following examples and experimental examples are provided in more detail. However, these examples and experimental examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples and experimental examples.

[0091]

[0092] Example 1: Method for isolating bovine or porcine muscle stem cells

[0093] To isolate muscle stem cells from bovine muscle tissue, the following experiments were performed.

[0094] First, bovine muscle tissue was washed with a solution containing 1% (v / v) AA (antibiotic-antimycotic) in Dulbecco's phosphate-buffered saline (DPBS). The washed tissue was cut into small pieces and washed again with a solution containing antibiotics. Subsequently, the tissue was digested by treating with enzymes (bromelain, papain, or pronase) at 37°C for a certain period of time. The enzymatically digested tissue was suspended in HG-DMEM (Dulbecco's Modified Eagle Medium, high glucose), washed, and centrifuged to collect the cell pellet. Subsequently, the cells were treated with red blood cell lysis agent at room temperature for 10 minutes, and the red blood cells were removed, and the cells were filtered sequentially using 100-, 70-, and 40-μm cell strainers. After the filtered cells were centrifuged, the supernatant was centrifuged again to collect the cell pellet, thereby obtaining cells derived from bovine muscle tissue.

[0095] Next, to recover muscle stem cells from 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 are 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. Thereafter, the medium was replaced with fresh medium every two days and cultured until the cell density reached 70-80%. The cultured cells were harvested using 0.25% trypsin-EDTA and used in subsequent experiments.

[0096] Porcine muscle stem cells were isolated in the same manner as bovine muscle stem cells, and the porcine muscle stem cell proliferation medium (pMuSC-PM) was composed of Ham's F10, 15% FBS, 1% AA, and 10 ng / mL rbFGF2.

[0097]

[0098] Example 2: Method for isolating bovine or porcine adipose stem cells

[0099] To isolate SVF (stromal vascular fraction), adipose stem cells, from bovine adipose tissue, the following experiments were performed.

[0100] First, bovine white adipose tissue was washed with a solution containing 1% (v / v) antibiotic-antimycotic and 10 mg / mL gentamicin in DPBS. The washed tissue was cut into small pieces and digested using an enzyme (collagenase or papain) at 37°C for 60 minutes. The enzymatically digested tissue was suspended in a medium (bovine SVF proliferation medium, bSVF-PM) containing 10% FBS, 1% antibiotic-antimycotic, and 10 mg / mL gentamicin in HG-DMEM, washed, and the suspension was filtered using a 100-μm cell strainer. After centrifugation, the cells were treated with red blood cell lysis agent for 1 minute at room temperature, and the red blood cells were filtered through a 40-μm cell strainer. After centrifugation again, the cell pellet was recovered, resuspended in bovine SVF growth medium, and cultured in a flask coated with 0.1% (w / v) fish gelatin. The culture was performed in a humidified environment of 37℃ 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 harvested by treatment with 0.25% trypsin-EDTA and used in subsequent experiments. Porcine SVF was isolated using the same method as bovine SVF isolation, and the porcine SVF growth medium (pSVF-PM) used a medium consisting of DMEM / F12, 10% FBS, 1% antibiotic-antimycotic, and 10 mg / mL gentamicin.

[0101]

[0102] Example 3: Cell culture method

[0103] Bovine and porcine muscle stem cells were cultured in T75 and T175 flasks, and each cell was recovered and cultured using DPBS, 0.25% trypsin-EDTA, and bovine muscle stem cell growth medium or porcine muscle stem cell growth medium. Cell proliferation was evaluated based on the PD (population doubling) value and cumulative growth cell number at each passage, and the cumulative growth cell number was calculated by multiplying the initial cell seeding number by the PDL (population doubling level). To analyze the purity and differentiation ability of the cells, bovine muscle stem cells were prepared by seeding 1 x 10^5 cells in a 24-well plate and 3 x 10^5 cells in a 6-well plate, respectively. After 2 days, the purity of the cells was confirmed using the prepared samples, and the cells were cultured for 4 days by replacing the differentiation medium (HG-DMEM, 1% penicillin-streptomycin, 2.5 mM insulin) to induce cell differentiation.

[0104] Bovine / porcine SVF were also cultured in T75 and T175 flasks, and cells were cultured using DPBS, 0.25% trypsin-EDTA, and bSVF-PM or pSVF-PM.

[0105]

[0106] Experimental Example 1: Determining the Optimal Papain Treatment Time for Muscle Stem Cell Isolation

[0107] To isolate muscle stem cells from bovine muscle tissue, papain was used as an enzyme, and the following experiments were performed to optimize the papain treatment time.

[0108] Specifically, based on the method described in Example 1, muscle stem cells were isolated from bovine muscle tissue, and papain was used at a concentration of 2000 units / ml as an enzyme, and after treatment for 30, 60, or 90 minutes, the total number of cells isolated from the tissue (isolated cells), adhesion ability, number of primary cultured cells (sorted cells, muscle stem cells), number of cell proliferation, and differentiation ability were confirmed. Meanwhile, the control group was treated with pronase, a commonly used enzyme, at a concentration of 2000 units / ml for 60 minutes.

[0109] As a result of the above experiment, the number of cells separated immediately after enzyme treatment was low when treated for 30 minutes, but it 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).

[0110] Next, the number of muscle stem cells (sorted cells) separated according to enzyme treatment was confirmed. When papain was treated for 30 minutes, muscle stem cells did not proliferate and could not be measured. However, when papain was treated for 60 minutes, it was confirmed that muscle stem cell separation efficiency was significantly superior compared to the 90-minute treatment or the control group (Fig. 3). In addition, when the separated muscle stem cells were continuously cultured, it was confirmed that when papain was treated for 60 minutes, the cumulative growth cell number was significantly superior compared to other experimental groups and the control group (Fig. 4).

[0111] Next, the differentiation ability of muscle stem cells separated by the above method was confirmed, and it was confirmed that myogenic differentiation was progressing in all experimental and control groups, and it was found that treatment with papain as an enzyme did not affect the cell's inherent characteristics (Fig. 5).

[0112] 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 separation efficiency.

[0113]

[0114] Experimental Example 2: Determining the optimal papain treatment concentration for muscle stem cell isolation (1)

[0115] To determine the optimal treatment concentration for isolating muscle stem cells from bovine muscle tissue using papain, the following experiments were performed.

[0116] 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 cells isolated from the tissue (isolated cells), the number of primary cultured cells (sorted cells, muscle stem cells), and differentiation potential were confirmed.

[0117] First, the number of cells isolated immediately after enzyme treatment was checked by treating with papain concentrations set to 200, 1000, 2000, or 5000 unit / mL. As a result, the number of isolated cells itself was small when treated with a concentration of 200 or 1000 unit / mL, and the number of isolated cells significantly increased when treated with a concentration of 2000 or 5000 unit / mL (Fig. 6). In addition, it was confirmed that the number of isolated cells was similar when treated with a papain concentration of 2000, 3000, 4000, or 5000 unit / mL (Fig. 7).

[0118] Next, the number of muscle stem cells (sorted cells) isolated by treating with papain at a concentration of 4000 or 5000 units / mL was confirmed, and it was confirmed that the muscle stem cell separation efficiency was significantly better than when treated with 2000 units / mL of FIG. 3 (FIG. 8). It was confirmed that muscle stem cells isolated under the above conditions progressed to myogenic differentiation, and it can be seen that treating with papain at the above concentration does not affect the cell-specific characteristics (FIG. 9).

[0119] Based on the above results, it can be seen that muscle stem cells can be effectively separated from muscle tissue when the papain treatment concentration is in the range of 2000 to 5000 unit / mL.

[0120]

[0121] Experimental Example 3: Determining the Optimal Papain Treatment Concentration for Muscle Stem Cell Isolation (2)

[0122] To further determine the optimal treatment concentration for isolating muscle stem cells from bovine muscle tissue using papain, the following experiments were performed.

[0123] 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 a concentration of 2000 or 20000 units / mL, the total number of cells isolated from the tissue (isolated cells), the number of primary cultured cells (sorted cells, muscle stem cells), and differentiation potential were confirmed.

[0124] First, the number of cells separated immediately after enzyme treatment was confirmed, and it was confirmed that the number of cells treated at a concentration of 20,000 units / mL was similar to that treated at a concentration of 2,000 units / mL (Fig. 10).

[0125] In addition, as a result of checking the number of separated muscle stem cells (sorted cells), it was confirmed that the muscle stem cell separation efficiency was significantly better than when treated with 2000 unit / mL of the above Fig. 3 (Fig. 11), and it was confirmed that muscle differentiation was progressing in the muscle stem cells separated under the above conditions, so it can be seen that when treating with the above concentration of papain, the cell-specific characteristics are not affected (Fig. 12).

[0126] Based on the above results, it can be seen that muscle stem cells can be effectively separated from muscle tissue even when the papain treatment concentration is 20,000 units / mL.

[0127]

[0128] Experimental Example 4: Determining the Optimal Papain Treatment Concentration for Muscle Stem Cell Isolation (3)

[0129] To further determine the optimal treatment concentration for isolating muscle stem cells from bovine muscle tissue using papain, the following experiments were performed.

[0130] 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 a concentration of 20,000 or 200,000 units / mL, the total number of cells isolated from the tissue (isolated cells), the number of primary cultured cells (sorted cells, muscle stem cells), and differentiation potential were confirmed.

[0131] First, the number of cells separated immediately after enzyme treatment was confirmed, and it was confirmed that the number of cells separated was significantly lower when treated at a concentration of 200,000 units / mL compared to when treated at a concentration of 20,000 units / mL, indicating that the cell separation efficiency was significantly reduced (Fig. 13).

[0132] In addition, as a result of checking the number of isolated muscle stem cells (sorted cells), it was confirmed that when treated with a concentration of 200,000 units / mL, the number of muscle stem cells was significantly lower compared to when treated with a concentration of 20,000 units / mL (Fig. 14), and it was confirmed that the muscle stem cells isolated by treating with a concentration of 200,000 units / mL also had a reduced differentiation ability (Fig. 15).

[0133] Based on the above results, it can be seen that when the papain treatment concentration is 200,000 units / mL, the efficiency of isolating muscle stem cells from muscle tissue is significantly reduced.

[0134]

[0135] Experimental Example 5: Identification of Muscle Stem Cells Isolated Using Papain

[0136] As confirmed through the above experimental example, the following experiment was performed to confirm whether muscle stem cells were effectively isolated from bovine muscle tissue using papain.

[0137] Specifically, based on the method described in Example 1, muscle stem cells were isolated from bovine muscle tissue, and 20,000 units / 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.

[0138] As a result of the above, it was confirmed that cells isolated from muscle tissue using papain expressed the PAX7 protein (Fig. 16), indicating that the isolated cells were muscle stem cells.

[0139]

[0140] Experimental Example 6: Confirming the Efficiency of Bromelain in Isolating Muscle Stem Cells

[0141] To determine whether muscle stem cells can be effectively isolated from muscle tissue using bromelain, an enzyme other than papain, the following experiments were performed.

[0142] Specifically, based on the method described in Example 1, we attempted to isolate muscle stem cells from bovine muscle tissue, and treated it with bromelain as an enzyme for 60 minutes, and confirmed the total number of cells isolated from the tissue (isolated cells) and the number of primary cultured cells (sorted cells, muscle stem cells) after treating it at a concentration of 2500, 5000, or 20000 units / mL. Meanwhile, the control group was treated with pronase, a commonly used enzyme, at a concentration of 2000 units / mL for 60 minutes.

[0143] As a result of the above experiment, it was confirmed that the number of isolated cells was significantly lower when bromelain was treated compared to when pronaase was treated (Fig. 17), and the muscle stem cell isolation efficiency was also significantly lower compared to the control group (Fig. 18).

[0144] Based on the above results, it can be seen that bromelain as an enzyme for isolating muscle stem cells from muscle tissue shows significantly low efficiency.

[0145]

[0146] Experimental Example 7: Evaluation of Papain for Isolating Porcine Muscle Stem Cells

[0147] To determine whether muscle stem cells can be isolated from porcine muscle tissue using papain, the following experiments were performed.

[0148] Specifically, muscle stem cells were isolated from pig muscle tissue based on the method described in Example 1, and 20,000 units / mL of papain as an enzyme was treated for 60 minutes, and cells isolated from the tissue were observed under a microscope.

[0149] 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 manner 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.

[0150]

[0151] Experimental Example 8: Evaluation of Papain's Adipose Stem Cell Isolation

[0152] To determine whether papain can be used to isolate stromal vascular fraction (SVF), a type of adipose stem cell, from adipose tissue, the following experiments were performed.

[0153] Specifically, based on the method described in Example 1, SVF was isolated from porcine adipose tissue, and 2500, 5000, or 10,000 units / mL of papain as an enzyme were treated for 60 minutes, and the residual tissue and cells isolated from the tissue after enzyme treatment were observed. Meanwhile, the control group was treated with collagenase 1, a commonly used enzyme, at a concentration of 0.1% for 60 minutes.

[0154] As a result of the above experiment, it was confirmed that adipose stem cells could be separated from adipose tissue, but the separation efficiency was lower than that of the control group, collagenase (Fig. 20).

[0155]

[0156] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A composition for separating stem cells, comprising papain.

2. A composition according to claim 1, wherein the composition is for isolating stem cells from animal tissue.

3. A composition according to claim 2, wherein the tissue is derived from at least one selected from the group consisting of chicken, bovine, and porcine.

4. A composition according to claim 2, wherein the tissue is derived from at least one selected from the group consisting of muscle and fat.

5. A composition according to claim 1, wherein the stem cells include at least one selected from the group consisting of muscle stem cells and adipose stem cells.

6. A composition according to claim 1, wherein the composition comprises 1,000 units / ml to 200,000 units / ml of papain.

7. A method for isolating stem cells, comprising a step of treating tissue separated from an individual other than a human with papain.

8. A method according to claim 7, wherein the step of treating the separated tissue with papain is performed for 30 to 90 minutes.

9. A method according to claim 7, wherein the step of treating the separated tissue with papain comprises treating papain at a concentration of 2,000 units / ml to 200,000 units / ml or a composition containing papain at the concentration.

10. A cell culture method comprising a step of culturing stem cells isolated using the method of any one of claims 7 to 9.

11. A method for producing cultured meat, comprising a step of culturing stem cells isolated using the method of any one of claims 7 to 9.

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