Method for establishing immortalized myoblast cell line, myoblast cell line, method for inducing differentiation into myotube cell-like cell, method for producing cultured meat, and cultured meat

Abstract

To provide a method for establishing an immortalized myoblast cell strain, by which the cloned immortalized myoblast cell strain derived from one cell can be obtained, to provide the myoblast cell strain, to provide a method for inducing the differentiation of a myotube cell-like cell, to provide a method for producing cultured meat, and to provide the cultured meat.SOLUTION: The method for establishing an immortalized myoblast cell line according to the present invention comprises a step of separating myoblasts from Japanese eel muscle tissue, a step of subjecting the separated myoblasts to primary culture in L-15 medium in the presence of fetal bovine serum (FBS) under conditions of 20 to 32 °C, a step of subjecting the myoblasts to single cell cloning at an early stage of the primary culture, and a step of culturing the myoblasts subjected to single cell cloning in L-15 medium in the presence of fetal bovine serum (FBS) under conditions of 20 to 32 °C, in this order.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a method for establishing an immortalized myoblast cell line, a method for inducing differentiation of a myoblast cell line, a method for producing myotube-like cells, a method for producing cultured meat, and cultured meat. [Background technology]

[0002] In recent years, there has been concern about food protein shortages, and cultured meat made from animal cells has attracted attention as a means of securing protein without relying on natural resources. In particular, Japanese eels have become so depleted that they have been designated an endangered species, and research into their sustainable use is urgently needed.

[0003] Traditionally, established muscle cell lines such as mouse C2C12 and rat L6 have been established in mammals, and intensive research into in vitro muscle development and differentiation has been conducted using these cell lines (see Non-Patent Document 1). It has also been shown that mouse myoblast C2C12 differentiates into myotubes in low serum conditions (see Non-Patent Document 2). Furthermore, with the aim of applying this to cultured meat, pluripotent stem cells (ES cells) derived from fertilized embryos of domestic cattle have been established, and it has already been shown that these ES cells can differentiate into muscle cells (see Non-Patent Document 3).

[0004] In addition, research into cultured meat is currently underway in mammals, birds, and other animals, and it is available for sale in some countries. However, the seed cells for cultured meat are currently derived from living organisms. Furthermore, in the case of fish, myoblast cells have already been established and sold in Atlantic mackerel (see Non-Patent Document 4). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Biochemical and Biophysical Research Communications 528 (2020) 193-198 [Non-patent document 2] Am. J. Physiol. Cell Physiol. 324: C420-C427, 2023 [Non-patent document 3] https: / / bio.nikkeibp.co.jp / atcl / news / p1 / 23 / 07 / 05 / 10883 / [Non-patent document 4] Scientific Reports 13, Article number: 5098 (2023) Summary of the Invention [Problem to be solved by the invention]

[0006] The seed cells of the above-mentioned myoblasts derived from Atlantic mackerel are currently derived from living organisms, and according to the inventors' investigations, there is a possibility that they are not cloned cells derived from a single cell, and that they may be contaminated with fibroblasts, etc., which has been a problem.

[0007] An objective of the present invention is to provide a method for establishing an immortalized myoblast cell line, which allows for the production of a cloned immortalized myoblast cell line derived from a single cell, a method for inducing differentiation of myoblast cell lines and myotube-like cells, a method for producing cultured meat, and cultured meat. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems, and as a result have discovered that single-cell cloning can be performed at the early stage of culturing Japanese eel myoblasts, allowing the establishment of an immortalized cell line derived from a single cell, thereby completing the present invention.

[0009] That is, the present invention is as follows. [1] 1. A method for establishing an immortalized myoblast cell line, comprising: a) isolating myoblasts from Japanese eel muscle tissue; b) primary culturing the isolated myoblasts in an L-15 medium in the presence of fetal bovine serum (FBS) at 20 to 32°C; c) performing single cell cloning on the myoblasts at an early stage of primary culture; d) culturing the myoblasts subjected to single cell cloning in L-15 medium in the presence of fetal bovine serum (FBS) at 20 to 32°C; A method comprising, in this order: [2] The method according to [1], wherein the single cell cloning at the early stage of primary culture in step c) is performed on muscle tissue-derived myoblasts of passages 1 to 4. [3] A myoblast cell line obtained by the method described in [1]. [4] This myoblast cell line is deposited at the National Patent Microorganism Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) (accession number: NITE P-04086). [5] A method for inducing differentiation of myotube-like cells from the myoblast cell line according to [3] or [4], comprising: e) culturing the myoblast cell line in L-15 medium (growth medium) in the presence of fetal bovine serum (FBS) at 20 to 32°C; f) inducing differentiation of the myoblast cell line into myotube-like cells by culturing the myoblast cell line in an L-15 medium (differentiation medium) in the presence of horse serum (HS) at 20 to 32°C; A method comprising, in this order: [6] A method for producing cultured meat from myotube-like cells induced to differentiate from the myoblast cell line described in [3] or [4], g) culturing the myoblast cell line in L-15 medium (growth medium) in the presence of fetal bovine serum (FBS) at 20 to 32°C; h) culturing the myoblast cell line in L-15 medium (differentiation medium) in the presence of horse serum (HS) at 20 to 32°C to induce differentiation into myotube-like cells to obtain cultured meat; A method of making the same, comprising the steps of: [7] Cultured meat obtained from the myoblast cell line described in [3] or [4]. [Effects of the Invention]

[0010] According to the present invention, it is possible to obtain a cloned immortalized myoblast cell line derived from a single cell, and it is possible to provide a method for establishing an immortalized myoblast cell line, a method for inducing differentiation of myoblast cell lines and myotube-like cells, a method for producing cultured meat, and cultured meat. Furthermore, by cloning and establishing a line of only myoblasts without killing living Japanese eels, an endangered species, and isolating the cells, it is possible to increase the number of stockpiled established myoblast lines and induce differentiation into myotube-like cells without them being replaced by fibroblasts or the like, and ultimately to produce Japanese eel cultured meat. [Brief explanation of the drawings]

[0011] [Figure 1A] This figure shows an enrichment analysis using Metascape of genes expressed in cell populations derived from Japanese eel muscle tissue. Comparing cell populations immediately after isolation with cell populations at the 8th passage, it shows the properties of a group of genes whose expression levels decrease by more than four-fold. [Figure 1B] This figure shows an enrichment analysis using Metascape of genes expressed in cell populations derived from Japanese eel muscle tissue. Comparing cell populations immediately after isolation with cell populations at the 16th passage, it shows the properties of a group of genes whose expression levels decrease by more than fourfold. [Figure 2] This figure shows the results of RT-qPCR quantification of muscle-related gene expression in cell populations derived from Japanese eel muscle tissue. In the figure, #01 represents a cell population immediately after isolation, #08 represents a cell population at passage 8, and #16 represents a cell population at passage 16 (n=2). [Figure 3]This figure shows the results of RT-qPCR quantification of lipid-related gene expression in cell populations derived from Japanese eel muscle tissue. In the figure, #01 represents the cell population immediately after isolation, #08 represents the cell population at passage 8, and #16 represents the cell population at passage 16 (n=2). [Figure 4] This figure shows cloned Japanese eel myoblasts (C4 clone cells) of the present invention. All scale bars in the figure indicate 100 μm. A and B show clones with myoblast-like morphology immediately after seeding, C and D show those in a confluent state, and E and F show spherical undifferentiated myoblasts and elongated fibrous myotubes detached from the bottom of the culture vessel, respectively. [Figure 5] 1 shows that the cloned Japanese eel myoblasts (C4 clone cells) of the present invention express myosin heavy chain, a muscle cell marker. The scale bar in the figure indicates 100 μm. [Figure 6] This figure shows the results of RT-qPCR quantification of muscle-related gene expression, comparing a cell population derived from Japanese eel muscle tissue of the present invention with cloned myoblasts (C4 clone cells). In the figure, #01 represents a cell population immediately after isolation, #08 represents a cell population at passage 8, #16 represents a cell population at passage 16, and C4 represents a cloned Japanese eel myoblast (n=6). [Figure 7] This figure shows that the cell population from Japanese eel muscle tissue of the present invention and myotube-like cells obtained by inducing differentiation of cloned Japanese eel myoblasts (C4 clone cells) (C4 differentiation induction (HS) and C4 differentiation induction (FBS)) express the muscle cell marker myosin heavy chain (Myh71). In the figure, #01 represents a cell population immediately after isolation, #08 represents a cell population at passage 8, #16 represents a cell population at passage 16, C4 represents cloned Japanese eel myoblasts, C4 differentiation induction (HS) represents C4 cells induced to differentiate by replacing the medium with a differentiation medium, and C4 differentiation induction (FBS) represents C4 cells induced to differentiate in the same medium (n=6). DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, an embodiment of the present invention will be described.

[0013] About immortalized cell lines The present invention relates to a method for establishing an immortalized myoblast cell line. An immortalized cell line refers to cultured cells that can grow indefinitely. On the other hand, normal cells have a limit to the number of cell divisions they can undergo and cannot divide indefinitely (usually, they cease to divide after 60 to 80 cell divisions).

[0014] Characteristics of fish-derived cells Fish cells, including Japanese eel cells, used in the present invention exhibit lifelong or indeterminate growth, whereas mammalian cells exhibit determinate growth, and it is extremely rare for mammalian cells isolated from normal tissue to become established and immortalized (usually, they cease to exist after 60-80 cell divisions). Furthermore, while a culture temperature of around 37°C is preferred for mammalian cells, a culture temperature of 20-30°C is preferred for fish cells. Furthermore, fish cells are considered to be relatively easy to immortalize, as they can grow even when left on a laboratory bench.

[0015] The preferred media for culturing fish cells are L-15 medium and a medium made by mixing equal parts of L-15 medium and DMEM / F12 medium, with L-15 medium being the most preferred. L-15 medium is cultured in the absence of CO2 (no CO2 incubator required), has a high amino acid concentration, contains L-gln, phenol red, and sodium pyruvate, and is commercially available from various reagent companies. For mammalian cell lines such as mouse C2C12 and rat L6 myoblast cells, DMEM medium in the presence of 10% fetal bovine serum (FBS) is used.

[0016] Isolation of myoblasts from muscle tissue (living Japanese eel) Myoblasts can be isolated from muscle tissue derived from living Japanese eels using known methods. In the present invention, a method based on the conventional methods of Kong et al. (Gene 2021, 802, 145869) and Froehlich et al. (Journal of Visualized Experiments 2014, 86, e51354) was used. Primary myoblasts obtained by treating living muscle tissue with a degrading enzyme (e.g., collagenase) can be used. Primary myoblasts may be filtered to remove impurities such as connective tissue.

[0017] Growth medium The growth medium in the present invention is most preferably L-15 medium containing 15% fetal bovine serum (FBS), a high-concentration serum, 1% antibiotic-antimycotic mixed solution, 1% MEM non-essential amino acid solution, and 10 ng / mL bFGF (basic fibroblast growth factor). The concentration of fetal bovine serum (FBS) added to the growth medium is preferably 8 to 17%, more preferably 9 to 15%, and most preferably 10 to 15%.

[0018] The growth medium of the present invention may or may not contain physiologically active substances, nutritional factors, or growth factors necessary for cell survival or proliferation. These factors may be added to the medium in advance or may be added during cell culture. The factors may be added during culture in any form, such as a single solution or a mixed solution of two or more solutions, and may be added continuously or intermittently.

[0019] Examples of physiologically active substances include insulin, IGF-1, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (bFGF, TGF-β, leukemia inhibitory factor (LIF), etc.), etc. Among these, the concentration of bFGF is preferably 0 to 10 ng / mL, more preferably 1 to 10 ng / mL, and most preferably 5 to 10 ng / mL.

[0020] Nutritional factors include sugars, amino acids, vitamins, hydrolysates, lipids, and the like.

[0021] Examples of sugars include glucose, mannose, and fructose, and these may be used alone or in combination of two or more.

[0022] Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and these may be used alone or in combination of two or more.

[0023] Examples of vitamins include D-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin, and DL-α-tocopherol, and these may be used alone or in combination of two or more. Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the growth medium of the present invention as needed.

[0024] About Single Cell Cloning In the present invention, single cell cloning refers to isolating a single cell from a primary cell population and producing a genetically identical monoclonal population in an isolated state from this single cell. Specifically, the single cell cloning refers to producing muscle tissue-derived myoblasts by isolating a single cell from a Japanese eel primary myoblast population at the early stage of primary culture. Single cell cloning is an important technique used for establishing cell lines in antibody drug research and development, regenerative medicine research, and the like. In the single cell cloning of the present invention, the number of passages of myoblasts after separation from primary cells is preferably 1 to 4, more preferably 1 to 3, and most preferably 1 to 2.

[0025] Differentiation medium The differentiation medium used herein most preferably uses L-15 medium as the basal medium. This L-15 medium is cultured in the absence of CO2 (no CO2 incubator required) and contains L-gln, phenol red, and sodium pyruvate. Furthermore, the differentiation medium is most preferably L-15 medium containing 2% horse serum (HS), a low-concentration serum, and 1% antibiotic-antimycotic mixed solution. The differentiation medium does not contain bFGF or MEM non-essential amino acid solution. The concentration of horse serum (HS) added to the differentiation medium is preferably 1-3%, more preferably 2-3%, and most preferably 2%.

[0026] The differentiation medium of the present invention may or may not contain physiologically active substances, nutritional factors, growth factors, and the like necessary for cell survival or proliferation. These factors may be added to the medium in advance or may be added during cell culture. The method of adding factors during culture may be in any form, such as one solution or a mixed solution of two or more solutions, and may be added continuously or intermittently.

[0027] Physiologically active substances include insulin, IGF-1, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (basic fibroblast growth factor (bFGF), TGF-β, leukemia inhibitory factor (LIF), etc.), etc.

[0028] Nutritional factors include sugars, amino acids, vitamins, hydrolysates, lipids, and the like.

[0029] Examples of sugars include glucose, mannose, and fructose, and these may be used alone or in combination of two or more.

[0030] Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and these may be used alone or in combination of two or more.

[0031] Examples of vitamins include D-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin, and DL-α-tocopherol, and these may be used alone or in combination of two or more.

[0032] Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the differentiation medium of the present invention as needed.

[0033] Culture and temperature conditions In the present invention, the culture temperature is preferably 20 to 32°C, more preferably 26 to 30°C, and most preferably 28°C, at all stages of the culture process, including after isolation of myoblasts from Japanese eel muscle tissue, the production of myoblast cell lines after single cell cloning, the induction of differentiation from myoblast cell lines to myotube-like cells, and the culture process from myotube-like cells to cultured meat.

[0034] Points to note from myoblast cell lines to myotube-like cells, muscle proteins, and cultivated meat As described in this example, when differentiating myoblasts into myotubes, it is usually preferable to change the growth medium containing a high concentration of serum to a differentiation medium with a low serum concentration (see, for example, Sci. Rep. 2023, 13, 5098). However, as shown in this example, the Japanese eel myoblast cell line C4 cells (JEM1129) of the present invention spontaneously differentiate into myotubes upon reaching confluence without changing the growth medium to a differentiation medium. This suggests that the cells may be cultured in growth medium alone, as indicated by increased expression of the myosin heavy chain gene, a marker gene for muscle differentiation. For routine muscle differentiation, it is preferable to culture the cells until they reach confluence as described above, and then continue culturing for one week without changing the medium.

[0035] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. [Example]

[0036] [Example 1] A method for isolating and primary culturing myoblasts from muscle tissue Myoblast isolation was performed according to the methods of Kong et al. (Gene 2021, 802, 145869) and Froehlich et al. (Journal of Visualized Experiments 2014, 86, e51354).

[0037] Japanese eel fry measuring approximately 10 cm and 1 g were anesthetized with ice, disinfected by spraying 70% ethanol on the body surface, and immediately killed by decapitation. Skeletal muscle tissue was isolated from the individual using tweezers and dissection scissors.

[0038] The isolated muscle tissue was cut into 1.5 mm pieces using dissection scissors in a PBS solution containing 5% antibiotic-antimycotic mixed solution (Nacalai Tesque). 3 The muscle tissue was then minced to approximately 1 / 4 of the original size. The tissue was centrifuged at 200 × g for 3 minutes at 4°C, and the supernatant was discarded. The minced muscle tissue was washed with fresh PBS containing a 5% antibiotic-antimycotic solution, and then centrifuged under the same conditions and the supernatant was discarded. This procedure was repeated four times.

[0039] Two milliliters of Leibovitz's L-15 medium (Fujifilm Wako Pure Chemical Industries) containing 0.2% collagenase type IV (Sigma-Aldrich) was added to the washed muscle tissue fragments and incubated at room temperature for 1 hour while rotating on a roller mixer. After the incubation, 40 μL of L-15 medium containing 5% trypsin (Sigma-Aldrich) was added and incubated at room temperature for an additional 20 minutes while rotating on a roller mixer. After the incubation, the tissue fragments were passed through a syringe equipped with an 18G needle and further minced. The tissue was centrifuged at 200 × g for 5 minutes at room temperature, and the supernatant was discarded. The pellet was washed with L-15 medium containing 5% antibiotic-antimycotic solution, then centrifuged at 200 × g for 5 minutes at room temperature, and the supernatant was discarded. The precipitate was added to L-15 medium (hereafter referred to as growth medium) containing 15% fetal bovine serum (FBS), 1% antibiotic-antimycotic mixture, 1% MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10 ng / mL bFGF (KAC). The cells were seeded onto a 6-well plate pre-coated with Easy iMatrix-511 (matrixome) and cultured at 28°C.

[0040] [Reference example 1] qPCR primer design and cell evaluation using the primers for muscle cell evaluation Cell populations immediately after isolation from Japanese eel muscle tissue and cell populations at passages 8 and 16 Genes whose expression levels decreased by more than fourfold were extracted and subjected to enrichment analysis. Muscle-related genes were significantly reduced at passage 8 (Figure 1A), but at passage 16, not only muscle-related but also lipid-related gene expression was reduced (Figure 1B). These results revealed that the cell population immediately after isolation contained not only muscle cells but also lipid-related genes (Figure 1B). These results suggest that the cell population immediately after isolation contained not only muscle cells but also adipocytes, and that the proportion of these cells decreased with passage. Based on these results, we selected seven qPCR primers for muscle cell evaluation (cadhelin 15 (cdh15) (SEQ ID NOs: 1 and 2), myosin heavy chain 7-like (myh7l) (SEQ ID NOs: 3 and 4), myod1 (myoD) (SEQ ID NOs: 5 and 6), tropomodulin 1 (tmod1) (SEQ ID NOs: 7 and 8), dual specificity phosphatase 27 (dusp27) (SEQ ID NOs: 9 and 10), paired box 7a (pax7a) (SEQ ID NOs: 11 and 12), myomaker (mymk) (SEQ ID NOs: 13 and 14), and six adipocyte-related primers (lipoprotein lipase (lipase: lpl) (SEQ ID NOs: 15 and 16), fatty acid synthase (fasn) (SEQ ID NOs: 17 and 18), EBP cholesterol delta-isomerase (ebp) (SEQ ID NOs: 19 and 20), 1-acylglycerol-3-phosphate dehydrogenase ( ... The primers used were O-acyltransferase 3 (agpat3) (SEQ ID NOs: 21 and 22), ELOVL fatty acid elongase 2 (elovl2) (SEQ ID NOs: 23 and 24), and fatty acid binding protein 1b, tandem duplicate 1 (fabp1b. 1) (SEQ ID NOs: 25 and 26) (Table 1). RT-qPCR analysis using these primers confirmed that the expression levels of the target genes decreased with increasing passage number, consistent with the RNA-Seq data (Figures 2 and 3).This strongly suggests that the RT-qPCR method using these primers is effective not only for evaluating muscle cells but also for evaluating fat cells in Japanese eels.

[0041] [Table 1]

[0042] [Example 2] A method for single cell cloning at the early stage of culturing a myoblast population The number of cells in the Japanese eel muscle tissue-derived primary myoblast population from about 1 to 3 passages described above in [Example 1] was counted, and the cell concentration was adjusted so that less than one cell was seeded per well, i.e., a cell concentration of about 0.5 cells / 100 μL, and 100 μL of each was seeded into a 96-well plate. The plate was observed over time, and cells in wells that showed sufficient proliferation were detached using trypsin and subcultured into a 24-well plate. After sufficient proliferation, the cell morphology was observed, and clones with a myoblast-like morphology, i.e., an elongated spindle-shaped morphology, were selected and subcultured into a 6-well plate. Further, the cells were transferred to T25 flasks and T75 flasks for expansion. The expanded clones reached 5.0 × 10 6 ~8.0×10 6 The cells were cryopreserved in Cell Reservoir One (Nacalai Tesque) at a concentration of approximately 1000 cells / mL and used in various experiments. This established Japanese eel myoblast cell line was named C4 cells (JEM1129) (deposited at the National Patent Microorganism Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE), accession number: NITE P-04086).

[0043] Culture conditions Japanese eel myoblast cell line C4 cells (JEM1129) were cultured in L-15 medium (growth medium) containing 15% fetal bovine serum (FBS), 1% antibiotic-antimycotic mixture, 1% MEM non-essential amino acid solution, and 10 ng / mL bFGF in flasks for adherent cells (regardless of manufacturer). When the cells proliferated and completely covered the adhesive surface of the culture vessel, i.e., when they reached confluence, they were subcultured. The medium was removed using an aspirator, and then washed with an appropriate amount of PBS(-) (Nacalai Tesque, Fujifilm Wako Pure Chemicals, etc.). 0.25 w / v% trypsin-1 mmol / L EDTA·4Na solution (Nacalai Tesque, Fujifilm Wako Pure Chemicals, etc.) was added to a 1 cm thick culture vessel. 2 After the cells were sufficiently detached, growth medium was added in an amount approximately four times the amount of trypsin solution added to stop the enzyme reaction, and approximately 1 / 8 of the trypsin solution was added to a flask containing fresh growth medium to subculture the cells.

[0044] Single-cell cloning was performed using cell populations immediately after isolation from Japanese eel muscle tissue, resulting in the isolation of several clonal cell types. Among these, clones with myoblast-like morphology were observed as individual, independent cells immediately after seeding (Fig. 4A and 4B). However, as the cells proliferated and covered the adhesive surface of the culture vessel, i.e., reached confluence, spontaneous cell fusion occurred, forming elongated myotubes (Fig. 4C and 4D). When the cell populations in this state were detached from the culture vessel using trypsin, spherical undifferentiated myoblasts and numerous elongated, fibrous myotubes were observed (Fig. 4E and 4F).

[0045] [Example 3] Analysis of gene expression of myosin heavy chain, cadherin 15, myoD, pax7a, etc. in myoblast cell lines ≪Immunohistological staining≫ The anti-myosin heavy chain antibody MF20 is a monoclonal antibody known to cross-react with fish skeletal myosin heavy chain (https: / / dshb.biology.uiowa.edu / MF-20). MF20 culture supernatant was purchased from DSHB, and immunostaining was performed using standard methods. Specifically, cells were cultured in slides and chambers (Watson) with a surface treated with Easy iMatrix-511 and differentiated into myotubes as described below. Cells were fixed with 4% paraformaldehyde in phosphate buffer (Fujifilm Wako Pure Chemical Industries) and permeabilized with 0.1% Triton-X100 (regardless of manufacturer) in PBS(-). After blocking with 3% bovine serum albumin (regardless of manufacturer) in PBS(-), the primary antibody solution was incubated with 1 / 50th the volume of MF20 culture supernatant in 1% BSA-PBS(-). After the reaction was complete, the cells were washed and then incubated with 0.1% BSA-PBS(-) containing 1 / 1000 of the amount of Anti-IgG (H&L), Mouse (Goat)-DyLight 549 (Rockland Immunochemicals) as a secondary antibody solution. After washing, the cells were stained for nuclei with 0.1% BSA-PBS(-) containing 5 μg / mL Hoechst 33342 (regardless of manufacturer) and observed under a fluorescent microscope. The cells were observed to maintain expression of the myosin heavy chain, a muscle cell marker gene (Figure 5).

[0046] <Gene expression analysis (RT-qPCR)> Basically, the analysis was carried out in the same manner as in [Reference Example 1] above. Cells were cultured in growth medium in 12-well plates until they reached approximately 80-90% confluence. Total RNA was extracted using ISOGEN II (Nippon Gene), and single-stranded cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (Toyobo). Cadhelin 15 (cdh15) (SEQ ID NOs: 1 and 2), myoD (SEQ ID NOs: 5 and 6), and paired box 7a (pax7a) (SEQ ID NOs: 11 and 12) genes were used as marker genes for myoblasts / muscle progenitor cells (source of marker genes: https: / / patents.google.com / patent / JP7000321B2 / ja). These gene sequences were extracted from the predicted protein-coding regions of the Japanese eel genome sequence reported by Wang et al. (GigaScience 2022, 11, giac120), and qPCR primers specific to each gene were designed using Primer BLAST (Table 1). GAPDH (SEQ ID NOs: 27 and 28) was selected as a reference gene for relative quantification (Fish Shellfish Immunol. 2018, 73, 288-296). Using the above single-stranded cDNA and primer set, qPCR reactions were performed using THUNDERBIRD® Next SYBR qPCR Mix (Toyobo), and the expression levels of each gene were quantified using the Ct method.

[0047] As shown in Figure 2, when comparing the expression levels of myoblast marker genes in cell populations immediately after isolation from Japanese eel muscle tissue and after the 8th and 16th passages, it is clear that the expression levels of these genes decrease significantly with each passage. However, in the cloned myoblast cell line, C4 cells (JEM1129), the expression levels of muscle cell marker genes are approximately 2-7 times higher than in the cell population immediately after isolation (Figure 6). This strongly suggests that the myoblast cell line cloned in this study is a pure myoblast.

[0048] [Example 4] Method for inducing differentiation of myoblast cell lines into myotube-like cells The Japanese eel myoblast cell line C4 cells (JEM1129) were differentiated into myotubes as follows. Specifically, cells were cultured in an appropriate culture vessel containing growth medium until they reached approximately 80-90% confluence. After removing the growth medium using an aspirator, L-15 medium containing 2% horse serum (HS) (regardless of manufacturer) and 1% antibiotic-antimycotic mixed solution (hereafter referred to as differentiation medium) was added. The differentiation medium was replaced with fresh medium every 2-3 days.

[0049] [Example 5] Method for analyzing gene expression in myotube-like cells This was essentially performed using the same method as the gene expression analysis (RT-qPCR) described in Reference Example 1 above. When differentiating myoblasts, as in this experiment, it is common practice to switch from a growth medium containing a high concentration of serum to a differentiation medium with a low serum concentration (see, e.g., Sci. Rep. 2023, 13, 5098). However, these cells spontaneously differentiated into myotubes upon reaching confluence without switching to a differentiation medium, as suggested by the approximately 10-fold increase in expression of the myosin heavy chain 7l gene, a marker gene for muscle differentiation, upon differentiation induction (Figure 7).

[0050] [Example 6] From myotube-like cells to cultivated meat The above [Example 1] to [Example 5] suggest that if the Japanese eel myoblast cell line C4 cells (JEM1129) are mass-cultured, it is theoretically possible to obtain muscle protein, and ultimately Japanese eel cultured meat, without using individual Japanese eels. [Industrial Applicability]

[0051] By mass-culturing the Japanese eel myoblast cell line C4 cells (JEM1129) of the present invention, it will be possible to obtain muscle proteins and cultured meat without killing and using individual Japanese eels, and ultimately to enable the stable production of cultured Japanese eel meat as an alternative to living Japanese eels, which are an endangered species.

Claims

1. 1. A method for establishing an immortalized myoblast cell line, comprising: a) isolating myoblasts from Japanese eel muscle tissue; b) primary culturing the isolated myoblasts in L-15 medium in the presence of fetal bovine serum (FBS) at 20 to 32°C; c) performing single cell cloning on the myoblasts at an early stage of primary culture; d) culturing the myoblasts subjected to single cell cloning in L-15 medium in the presence of fetal bovine serum (FBS) at 20 to 32°C; A method comprising, in this order:

2. The method according to claim 1, wherein the single cell cloning at the initial stage of primary culture in step c) is performed on muscle tissue-derived myoblasts at passages 1 to 4.

3. A myoblast cell line, characterized in that it is obtained by the method of claim 1.

4. A myoblast cell line deposited at the National Patent Microorganism Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE) (accession number: NITE P-04086).

5. A method for inducing differentiation of myotube-like cells from the myoblast cell line according to claim 3 or 4, comprising: e) culturing the myoblast cell line in L-15 medium (growth medium) in the presence of fetal bovine serum (FBS) at 20 to 32°C; f) inducing differentiation of the myoblast cell line into myotube-like cells by culturing the myoblast cell line in L-15 medium (differentiation medium) in the presence of horse serum (HS) at 20 to 32°C; A method comprising, in this order:

6. A method for producing cultured meat from myotube-like cells induced to differentiate from the myoblast cell line according to claim 3 or 4, comprising: g) culturing the myoblast cell line in L-15 medium (growth medium) in the presence of fetal bovine serum (FBS) at 20 to 32°C; h) culturing the myoblast cell line in L-15 medium (differentiation medium) in the presence of horse serum (HS) at 20-32°C to induce differentiation into myotube-like cells, thereby obtaining cultured meat; A method of making the same, comprising, in this order:

7. Cultured meat, characterized in that it is obtained from the myoblast cell line described in claim 3 or 4.

Images