Method for producing satellite cells

Abstract

Provided is a method for producing satellite cells. The present invention uses a production method including (1) a step for performing an enzymatic treatment on a muscle, and (2) a step for combing the enzymatically-treated muscle by using a comb in a manner parallel to the direction of muscle fibers.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to Japanese Patent Application No. 2020-20778, filed on February 10, 2020, the entire disclosure of which is incorporated herein by reference. [Technical field]

[0002] The present invention relates to a method for producing satellite cells. [Background technology]

[0003] In order to produce beef steak meat by culturing, it is necessary to grow a large number of satellite cells (also called "satellite cells") and produce myotubes (muscle fibers). Conventionally, satellite cells were obtained by cutting skeletal muscle tissue into small pieces, dissociating the cells with digestive enzymes, and filtering the tissue to remove impurities such as cell debris (see, for example, Patent Document 1). However, this conventional method was highly contaminated with impurities such as fibroblasts, vascular endothelial cells, and adipocytes, and it was difficult to collect a fraction (a fraction containing satellite cells) that was rich in satellite cells and had reduced other impurities. In addition, a cell sorter or the like must be used to isolate satellite cells. Cell sorters and antibodies are expensive, and a process of washing the antibodies is required, so it was practically impossible to isolate a large number of satellite cells. In particular, for commercialization, it is necessary to efficiently obtain a large number of satellite cells in a short period of time.

[0004] In addition, methods for inducing skeletal muscle progenitor cells have been known, such as a skeletal muscle progenitor cell inducer consisting of the MyoD gene, the Pax7 gene and / or the Pax3 gene, and the Mef2b gene (see Patent Document 2), but no method for efficiently obtaining large amounts of satellite cells in a short period of time has been known. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-194 A [Patent Document 2] JP 2018-11546 A Summary of the Invention

[0006] The inventors have focused on the fact that skeletal muscle fibers run in parallel, and discovered that by treating satellite cells attached to the outside of muscle fibers with digestive enzymes and then combing them in two directions in parallel with the running of muscle fibers, it is possible to efficiently produce a large amount of satellite cells in a short period of time. The present invention is based on these findings.

[0007] Thus, the present invention discloses a method for producing satellite cells, comprising the steps of enzymatically treating muscle and combing the enzymatically treated muscle in parallel with the course of muscle fibers.

[0008] According to the present invention, the following inventions are provided. [1] A method for producing satellite cells, comprising the steps of: (1) enzymatically treating muscle; and (2) The process of combing the enzyme-treated muscle in parallel with the course of the muscle fibers. [2] The manufacturing method described in [1], which comprises repeating the steps of (1) enzymatically treating the muscle and (2) combing the enzyme-treated muscle in parallel with the course of the muscle fibers at least once. [3] The method according to [1] or [2], wherein the comb has at least one comb tooth. [4] The method according to any one of [1] to [3], wherein the muscle is a muscle of a mammal, a reptile, a bird, or a fish. [5] The method for producing a comb according to any one of [1] to [4], wherein the thickness of the comb teeth is 1 mm or less. [6] The manufacturing method according to any one of [1] to [5], wherein the tips of the comb teeth are shaped like blades. [7] The method according to [5] or [6], wherein the ratio of the length of the tips of the comb teeth to the length of the comb teeth (length of the tips of the comb teeth:length of the comb teeth) is 1:20 to 1:2. [8] A satellite cell produced by the production method described in any one of [1] to [7]. [9] A method for purifying satellite cells, comprising the steps of: (1) enzymatically treating muscle; and (2) The process of combing the enzyme-treated muscle in parallel with the course of the muscle fibers.

[10] Satellite cells purified by the purification method described in [9].

[11] A method for producing meat, comprising a step of culturing satellite cells produced by the production method described in any one of [1] to [7] or satellite cells purified by the purification method described in [9], and inducing them to become muscle fibers.

[12]

[11] Meat produced by the manufacturing method described in the above.

[0009] The method for producing satellite cells of the present invention is advantageous in that satellite cells can be produced efficiently in large quantities in a short period of time. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a perspective view of one embodiment of a comb used in the manufacturing method of the present invention. [Diagram 2] FIG. 1 is a diagram showing an outline of the configuration of one embodiment of a comb used in the manufacturing method of the present invention. [Diagram 3] FIG. 2 is a diagram showing the appearance of comb teeth of one embodiment of a comb used in the manufacturing method of the present invention. [Figure 4] 1 shows a microscopic photograph of a cell suspension containing satellite cells obtained by a conventional method (conventional method). [Diagram 5] 1 shows a microscopic photograph of a cell suspension containing satellite cells obtained by the comb method of the present invention. [Figure 6] The photograph shows a mass of muscle cells (colored with food coloring) transferred to a culture dish. [Figure 7]This shows an HE stained image of a paraffin section of a muscle cell cluster. Description of the Invention

[0011] [Method of producing satellite cells] According to one embodiment of the present invention, there is provided a method for producing satellite cells, comprising (1) a step of enzymatically treating muscle, and (2) a step of combing the muscle fibers of the enzyme-treated muscle in parallel with the running of the muscle fibers. The muscle used in the production method of the present invention may be any muscle, but may be, for example, muscle of mammals such as cows, pigs, humans, sheep, rabbits, mice, and whales, reptiles such as crocodiles, birds such as chickens, or fish such as tuna. Among these, the muscle of cows, pigs, humans, sheep, whales, rabbits, crocodiles, tuna, or chickens is preferable. When cows' muscles are used in the production method of the present invention, the obtained satellite cells can be used to produce steak meat, minced meat, etc., and when human muscles are used, the obtained satellite cells can be used as a cell source for treating myodegenerative diseases such as Duchenne muscular dystrophy (DMD).

[0012] The muscle site used in the manufacturing method of the present invention is not particularly limited, but is preferably the diaphragm, iliopsoas muscle, masseter muscle, gluteal muscle, or the like, and it is preferable to use a method for collecting the muscle that is free of contamination by bacteria, Bacillus subtilis, etc.

[0013] The enzyme used in the enzymatic treatment in the production method of the present invention may be any enzyme that can soften the muscle and make it easier to comb, but examples thereof include proteases, preferably one or more selected from the group consisting of collagenase, dispase, and trypsin, and more preferably a combination of collagenase and dispase.

[0014] The enzyme treatment in the production method of the present invention may be carried out in any manner as long as the muscle can be treated with the enzyme. For example, the enzyme treatment may be carried out by applying the enzyme to the muscle surface with a pipette or by immersing the muscle in the enzyme.

[0015] The comb used in the manufacturing method of the present invention may be of any type as long as it can plow through muscles, but is preferably a comb having at least one or more teeth (in this specification, even a comb with one tooth is included in the "comb" used in the manufacturing method of the present invention), more preferably a comb having 5 to 20 teeth, and even more preferably a comb having 5 to 10 teeth. The spacing between the teeth is not particularly limited, but is preferably equal, and the spacing between the teeth is, for example, 0.5 to 2 mm.

[0016] The material of the comb used in the manufacturing method of the present invention may be of any type as long as it is capable of plowing through muscles, but it is preferable that the material be capable of repeated sterilization and has the strength to withstand plowing through muscles, and it is more preferable that it be stainless steel or a titanium alloy, and it is particularly preferable that it be a titanium alloy.

[0017] The thickness of the teeth of the comb used in the manufacturing method of the present invention may be any thickness as long as it can plow through the muscle, but is preferably 1 mm (1000 microns) or less, and more preferably 0.05 mm to 1 mm. Here, the thickness of the teeth refers to the thickness of the teeth in the direction perpendicular to the course of the muscle fibers of the muscle tissue.

[0018] The shape of the teeth of the comb used in the manufacturing method of the present invention may be any shape as long as it can plow the muscle, and the shape of the tips of the teeth and the part other than the tips of the teeth may be the same or different, but it is preferable that they are different. The shape of the teeth of the comb may be convex (including, for example, the shape of a sword), concave, or flat without unevenness, but it is preferable that the tips of the teeth of the comb are in the shape of a blade, and it is more preferable that the tips of the teeth of the comb are in the shape of a blade and the part other than the tips of the teeth of the comb are flat without unevenness. By making the tips of the teeth of the comb used in the manufacturing method of the present invention into the shape of a blade, the combing ability when plowing the muscle is significantly improved. In another preferable embodiment, the shape of the teeth of the comb may be such that after plowing the muscle with the tips of the teeth of the comb that are in the shape of a blade, the muscle may be plowing with the tips of the teeth of the comb that are not in the shape of a blade (for example, flat without unevenness).

[0019] In the comb used in the manufacturing method of the present invention, when the tips of the comb teeth have different shapes from the parts other than the tips of the comb teeth, for example, when the tips of the comb teeth have a blade shape and the parts other than the tips of the comb teeth have a flat shape without any irregularities, the ratio of the length of the tips of the comb teeth to the length of the comb teeth (the sum of the length of the tips of the comb teeth and the length of the parts other than the tips of the comb teeth) (length of the tips of the comb teeth:length of the comb teeth) is preferably 1:20 to 1:2, more preferably 1:5 to 1:10. The length of the tips of the comb teeth is not particularly limited, but is, for example, 0.01 to 10 cm, preferably 0.1 to 3 cm, more preferably 0.3 to 1 cm.

[0020] In the manufacturing method of the present invention, the term "parallel" in the "step of combing in parallel to the course of the muscle fibers" means that the course of the muscle fibers and the direction of the comb are aligned, and does not have a strict meaning such as the course of the muscle fibers and the direction of the comb never intersecting, no matter how much they are extended.

[0021] One embodiment of the production method of the present invention is as follows. (i) Obtain a diaphragm from a slaughterhouse. (ii) The diaphragm was soaked in collagenase-dispase solution (37°C) for 30 minutes and then placed on a slanted tray with the diaphragm clamped with clips. (iii) Use a comb (which has 10 blades spaced 1 mm apart at the tip only; the number of blades can be changed as needed) to comb along the course of the muscle fibers so that the comb blades get evenly between the muscle fibers. (iv) The tip of a pipette is inserted between muscle fibers and the satellite cells are released by strongly spraying Hanks' balanced salt solution onto the muscle fibers. (v) The Hanks' solution containing the satellite cells is collected and centrifuged. (vi) Repeat the above steps (ii) to (v) 4 to 7 times. In addition, the satellite cells with few impurities obtained in the above steps (i) to (vi) can be grown themselves using atelocollagen beads or the like as described in the Examples below to produce a method for producing satellite cells. Therefore, the method for producing satellite cells of the present invention may further include a step of growing satellite cells.

[0022] Hereinafter, an embodiment of a comb used in the manufacturing method of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view of an embodiment of a comb used in the manufacturing method of the present invention. The comb shown in FIG. 1 is composed of a titanium metal plate including comb teeth 2, a titanium metal spacer 4 sandwiched between two metal plates, and a bolt connecting the titanium metal plate and the spacer 4, but the number of titanium metal plates and spacers is not limited to this. In addition, the length of the comb teeth 2 means the length of the titanium metal plate including the comb teeth 2 minus the length of the spacer 4. The length of the comb teeth 2 includes the blade 1. In addition, the length of the spacer 4 is the length of the body 3.

[0023] Fig. 2 is a diagram showing an outline of the configuration of one embodiment of a comb used in the manufacturing method of the present invention. Fig. 2 shows a diagram in which the bolts are removed from the comb in Fig. 1, and a titanium metal plate including comb teeth 2 and a spacer 4 are placed on a flat desk, and the titanium metal plate including comb teeth 2 and the spacer 4 are included. The number of titanium metal plates and spacers is not limited to this.

[0024] 3 is a diagram showing the appearance of a titanium metal plate including comb teeth of one embodiment of a comb used in the manufacturing method of the present invention. The thickness 5 of the comb teeth is in a direction perpendicular to the direction of the muscle fibers of the muscle tissue.

[0025] As described above, according to another aspect of the present invention, there is provided a satellite cell produced by the above-mentioned production method of the present invention. The satellite cell produced by the production method of the present invention is preferable in that it contains few impurities.

[0026] [Satellite cell purification method] According to another aspect of the present invention, there is provided a method for purifying satellite cells, comprising: (1) a step of enzymatically treating muscle; and (2) a step of combing the muscle fibers of the enzyme-treated muscle in parallel with the running of the muscle fibers. The muscle, enzyme, comb, etc. used in the method for purifying satellite cells may be the same as the muscle, enzyme, comb, etc. used in the method for producing satellite cells of the present invention. The satellite cells obtained by the method of the present invention contain significantly fewer impurities than those obtained by conventional methods, and therefore the method can also be regarded as a method for purifying satellite cells.

[0027] According to another aspect of the present invention, there is provided a satellite cell purified by the above-mentioned purification method of the present invention.

[0028] [Meat production method] According to another aspect of the present invention, there is provided a method for producing meat, comprising a step of culturing satellite cells produced by the above-mentioned production method of the present invention or satellite cells purified by the above-mentioned purification method of the present invention and inducing them to become muscle fibers.

[0029] Satellite cells are endogenous stem cells present in skeletal muscles and essential for the growth and regeneration of skeletal muscles. They usually exist in a resting state (G0 phase), but when a specific stimulus is applied, such as when skeletal muscles are damaged by injury, they are immediately activated and proliferate, and then differentiate into myoblasts to form new muscle fibers. In the meat production method of the present invention, any method can be used as long as it is possible to culture satellite cells and induce them into muscle fibers. For example, by adding a muscle satellite cell activating factor (e.g., a secretory factor such as a growth factor or cytokine, or nitric oxide) to the culture medium and culturing the satellite cells, the satellite cells can be activated and proliferated, and differentiated into myoblasts to induce muscle fibers. In addition, preferred examples of growth factors used as muscle satellite cell activating factors include growth hormone (GH), hepatocyte growth factor (HGF), steroids such as testosterone and dexamethasone, and insulin-like growth factor (IGF), and among these, testosterone is particularly preferred. In addition, preferred examples of cytokines used as muscle satellite cell activating factors include leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). In particular, it is preferable to culture satellite cells in a culture medium containing LIF, since they can proliferate in a satellite cell or myoblast state without forming myotubes (if satellite cells become myoblasts during culture and fuse with each other to form myotubes, they will not proliferate). Preferred methods for culturing satellite cells and inducing them into muscle fibers include a method in which satellite cells are converted into myoblasts using atelocollagen beads or the like, and then converted into muscle fibers using a steroid such as testosterone.

[0030] The culture conditions for satellite cells may be any conditions that allow induction into muscle fibers, but muscle fibers can be induced, for example, by culturing at 30 to 38° C. for 2 to 10 days.

[0031] Furthermore, as described in the Examples below, satellite cells obtained by the comb method of the present invention can be fractionated into satellite cell fractions and non-satellite cell fractions using a cell sorter device (such as a FACS device) based on flow cytometry, based on surface markers such as CD31, CD45, CD56, and CD29, and the non-satellite cell fraction can be further separated into a fibroblast fraction, a myoblast fraction, an endothelial cell fraction, etc., to efficiently induce cell masses derived from each type of cell.

[0032] [meat] According to another aspect of the present invention, there is provided meat produced by a meat production method comprising a step of culturing satellite cells produced by the above-mentioned production method of the present invention or satellite cells purified by the above-mentioned purification method of the present invention and inducing them into muscle fibers. The produced meat can be used, for example, as steak meat, minced meat, etc. EXAMPLES

[0033] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0034] Test Example 1: Comparative study of the recovery efficiency of satellite cells from bovine masseter muscles (1) Conventional method Masseter muscle collected from a cow was washed with Hanks' balanced salt solution (Nissui Pharmaceutical Co., Ltd.), and the washed meat chunks (approximately 10 g) were cut into small pieces with a scalpel and dissociated with digestive enzymes (collagenase solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and dispase solution (Thermo Fisher Scientific Co., Ltd.)) at 37°C. The dissociated solution was then filtered through a filter (a 100 μm pore size filter (Greiner Japan Co., Ltd.) and then a 30 μm pore size filter (pluriSelect Life Science UG&Co.KG)), and the solution was centrifuged (1500 rpm) for 5 minutes, and the number of satellite cells after centrifugation was counted using a cell counting board.

[0035] (2) The combing method of the present invention Masseter muscle taken from a cow was washed with Hank's balanced salt solution (manufactured by Nissui Pharmaceutical Co., Ltd.), and the washed meat chunks (400-1000 g) were immersed in digestive enzymes (collagenase solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and dispase solution (manufactured by Thermo Fisher Scientific Co., Ltd.)) for 30 minutes for enzyme treatment. The muscle chunks after enzyme treatment were combed, and the spaces between the muscle fibers were washed with Hank's balanced salt solution using a pipette, and the washing solution was collected. The collected washing solution was then filtered through a filter (100 μm pore size filter; manufactured by Greiner Japan Co., Ltd.), and then further filtered through a 30 μm pore size filter (manufactured by pluriSelect Life Science UG&Co.KG), and the liquid that passed through was centrifuged (1500 rpm) for 5 minutes. This series of operations from the enzyme treatment was repeated 4-5 times, and the number of satellite cells was counted using a cell counting board. The comb used in the combing method of the present invention was the comb shown in Figs.

[0036] (3) Measurement results of satellite cell count Using the above (1) conventional method, the number of satellite cells was counted at 64 per small piece of meat (approximately 10 g). On the other hand, in the above (2) combing method of the present invention, the meat mass (approximately 10 g) was 6 × 10 4 The number of satellite cells was counted. These results demonstrated that the recovery rate of satellite cells per meat loaf was approximately 1000 times higher with the comb method of the present invention than with the conventional method. Furthermore, when the cell suspension containing satellite cells obtained by the normal method (conventional method) in (1) above in Test Example 1 was observed under a microscope, it was difficult to find the satellite cells buried in a large amount of impurities, as shown in the photograph in Figure 4. On the other hand, in the cell suspension containing satellite cells obtained by the comb method of the present invention in (2) above in Test Example 1, many satellite cells were observed, as shown in the photograph in Figure 5, and it was found that there were significantly fewer impurities other than satellite cells. Furthermore, in the conventional method, the process of shredding a large amount of meat with a razor scalpel is very time-consuming, and it takes more than 12 hours to perform the series of operations in the conventional method and measure the number of satellite cells.However, in the comb method of the present invention, even if the series of operations is repeated 4 to 5 times, it can be completed in about 3 to 3.5 hours, thereby reducing the operation time to about 1 / 4 and significantly improving work efficiency.

[0037] Test Example 2: Analysis using flow cytometry A cell suspension of satellite cells obtained from the cheek meat collected from a cow by the same method as the comb method of the present invention in (2) of the above-mentioned Test Example 1 was divided into two, one of which was analyzed for the cell type and cell number distribution contained in the cell suspension using a cell analyzer by flow cytometry, and the other was analyzed by a cell analyzer by flow cytometry after 7 days of stirring culture. The CD31(-), CD45(-), CD56(+), and CD29(+) fractions were defined as satellite cell fractions, the ER-TR7(+) fraction of the non-satellite cell fractions was defined as the fibroblast fraction, the H36(+) fraction of the non-fibroblast fractions was defined as the myoblast fraction, and the Tie-2 / Tek(+) fraction of the non-myoblast fractions was defined as the endothelial cell fraction. The number of cells in each fraction was counted. The results are shown in Table 1 below. In addition, the cell suspension obtained by the comb method of the present invention had few impurities and was therefore suitable for flow cytometry analysis, whereas the cell suspension obtained by the conventional method had many impurities and was therefore not suitable for flow cytometry analysis. [Table 1]

[0038] From the results of Table 1 above, it was found that the cells obtained by the comb-plow method of the present invention are rich in satellite cells, are purified with few other impurities, and have the proliferation ability of any of the cells, satellite cells, myoblasts, fibroblasts, and endothelial cells, among which the proliferation ability of satellite cells is the highest. Therefore, it was found that the cells obtained by the comb-plow method of the present invention contain a large amount of satellite cells with sufficient proliferation ability for commercializing meat production.

[0039] Test Example 3: How to make minced meat From the cheek meat taken from a cow, 1 x 10 7 Satellite cells and atelocollagen beads (diameter 300-400 μm, 1x10 6 pieces), 1x10 5 10 vascular endothelial cells and atelocollagen beads (diameter 300-400 μm, 1x10 4 pcs) and 1x10 4 fibroblasts and atelocollagen beads (diameter 300-400 μm, 1x10 3The three beads were placed separately in three 10 cm petri dishes, and each was cultured with 10 ml of the growth medium described below. After 24 hours, the three were mixed in one agitator culture vessel, and placed in a culture medium containing 100 ml of growth medium (DMEM / F12 mixed with 15% FBS, 100 μM glutaMAX, 0.1% minimum essential medium (MEM)-nonessential amino acids (NEAAs), 50 U / ml penicillin, 50 μg / ml streptomycin, and 0.25 μg / ml fungizone) (all reagents were obtained from Thermo Fisher Scientific). The oxygen concentration was set to 5%, and the agitation culture was performed at 20 rpm to promote the proliferation of satellite cells. Four days after the start of the culture, 100 ml of new growth medium was added, and the culture was continued under the same conditions. The satellite cells attached to the surface of the beads proliferated and came to cover the surface of the beads (called satellite beads). On the 8th day of culture, in order to grow the satellite cells attached to the beads into myoblasts, 200 ml of culture medium containing 50 ng / ml testosterone (Fujifilm Wako Pure Chemicals, testosterone propionate, 208-17533) was added to the above culture medium, and the medium was cultured with stirring at 4.7% CO2 / air and 50 rpm. The cells that proliferated on the surface of the beads and became myoblasts (called myoblast beads) adhered to each other, forming large clumps of myoblast beads. When changing the culture medium, the stirring was stopped for 10 minutes, the clumps of cell beads were allowed to settle, 200 ml of the supernatant was removed, 200 ml of new culture medium containing testosterone was added, and culture was resumed at 50 rpm. The culture medium was changed twice a week. After two weeks of culture in the culture medium containing testosterone, the myoblasts grew into muscle cells and formed myotubes, so the culture was terminated at that point. Figure 6 shows a photograph of a mass of muscle cells (minced meat) cultured and produced in a stirred incubator, dyed with food coloring and then transferred to a 10 cm petri dish. The minced meat in Figure 6 was 10 cm in diameter, approximately 10 mm thick, and weighed approximately 80 grams. In addition, this mass of muscle cells (minced meat) was cut into paraffin sections and stained with HE to observe the tissue (see Figure 7). In this HE stained image, the striated (sarcomere) structure, which is characteristic of muscle, was clearly observed, and it was found that meat had been formed. From the above, it was found that meat can be produced by using the method for producing satellite cells of the present invention or the method for purifying satellite cells of the present invention. [Industrial Applicability]

[0040] The satellite cells produced by the production method of the present invention can be used to produce beef steak meat, etc. by culturing. [Explanation of symbols]

[0041] 1. Blade 2. Comb teeth 3. Torso 4. Spacer 5. Thickness of the teeth

Claims

1. A method for producing satellite cells, comprising the steps of: (1) enzymatically treating muscle; and (2) The process of combing the enzyme-treated muscle in parallel with the course of the muscle fibers.

2. 2. The method according to claim 1, wherein the steps of (1) enzymatically treating the muscle and (2) combing the muscle fibers in parallel with the course of the muscle fibers of the enzyme-treated muscle are repeated at least once.

3. 3. The method according to claim 1 or 2, wherein the comb has at least one comb tooth.

4. The method according to any one of claims 1 to 3, wherein the muscle is mammalian, reptilian, avian or fish muscle.

5. The method according to any one of claims 1 to 4, wherein the thickness of the comb teeth is 1 mm or less.

6. The method according to any one of claims 1 to 5, wherein the tips of the teeth of the comb are shaped like blades.

7. The method according to claim 5 or 6, wherein the ratio of the length of the tips of the comb teeth to the length of the comb teeth (length of the tips of the comb teeth:length of the comb teeth) is 1:20 to 1:

2.

8. A method for purifying satellite cells, comprising the steps of: (1) enzymatically treating muscle; and (2) The process of combing the enzyme-treated muscle in parallel with the course of the muscle fibers.

9. A meat production method comprising a step of culturing satellite cells produced by the production method according to any one of claims 1 to 7 or satellite cells purified by the purification method according to claim 8, and inducing them to become muscle fibers.

Images