Method for producing cultured meat

Abstract

To solve the problem in which: cultured meat requires effective proliferation of myoblasts; although serum-containing media are used for culturing, biologically derived serum is expensive and poses a risk of contamination due to viruses; on the other hand, serum-free media cause myoblasts to differentiate into myotubes, resulting in reduced cell proliferation.SOLUTION: There is provided a method for producing cultured meat by culturing myoblasts in a low-serum medium containing vitronectin. The low-serum medium can suppress rapid differentiation of myoblasts, which have proliferative ability, into myotubes, which lack proliferative ability, thereby enabling efficient proliferation of cells. Furthermore, cells can be effectively proliferated even in three-dimensional culture, and thus the method is suitably applicable to cultured meat production.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing cultured meat using a low-serum medium. [Background technology]

[0002] Cultured meat is made by culturing myoblast cells extracted from animals (derived from vertebrates such as mammals, birds, reptiles, amphibians, and fish). When cultured meat is put into practical use, it will eliminate the need to slaughter vertebrates for meat such as pigs, cows, chickens, and fish, reducing sacrifices. It will also reduce the risk of economic burden on producers by avoiding mass culling due to disease.

[0003] Patent Document 1 discloses a method for culturing cells, the method including a step of culturing cells in a serum-free medium containing one or more extracts selected from the group consisting of a bovine muscle tissue extract, a porcine muscle tissue extract, a chicken muscle tissue extract, and a white fish muscle tissue extract. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2023-93403 A Summary of the Invention [Problem to be solved by the invention]

[0005] Cell culture usually contains serum components such as fetal bovine serum. However, such serum is expensive and the composition ratio is not stable depending on the individual from which it is collected. Furthermore, since it is derived from a living body, the possibility of contamination by viruses, etc. cannot be denied. Therefore, as in Patent Document 1, serum-free media have been considered, but there is a problem that myoblasts rapidly differentiate into myotube cells in serum-free media, making cell proliferation itself difficult. [Means for solving the problem]

[0006] The present invention has been conceived in view of the above problems, and was achieved by discovering that vitronectin, a type of cell matrix, has the ability to inhibit cell differentiation.

[0007] Specifically, the method for producing cultured meat according to the present invention includes: The method is characterized by culturing myoblasts in a low-serum medium containing vitronectin. Effect of the Invention

[0008] By adding vitronectin, the present invention can suppress the differentiation of myoblasts into myotubes even in a low-serum medium, effectively promoting cell proliferation, which has the effect of allowing cultured meat to be cultivated to a predetermined size in a short period of time. [Brief description of the drawings]

[0009] [Figure 1] This is a graph showing the ratio of MHC positive cells to the total cell number under low serum culture conditions, using plate culture coated with various cell matrices at 0.5 to 2.5 μg / cm2. [Diagram 2] 2 is a graph showing the results of examining the cell count in the case of FIG. 1. [Diagram 3] These are micrographs of the fluorescent detection of MHC, a myotube cell marker protein, in the cases of the control and vitronectin in Figure 1. Figure 3(a) shows the control, and Figure 3(b) shows vitronectin. These are also micrographs of the cells after nuclear staining. Figure 3(c) shows the control, and Figure 3(d) shows vitronectin. [Figure 4] These are photographs showing the cell proliferation effect of vitronectin when mouse myoblasts and chicken myoblasts were cultured in 3D. Figure 4(a) shows the control for mouse myoblasts, Figure 4(b) shows mouse myoblasts with the addition of vitronectin, Figure 4(c) shows the control for chicken myoblasts, and Figure 4(d) shows chicken myoblasts with the addition of vitronectin. [Diagram 5]1 is a graph showing the concentration dependency of vitronectin on MHC positive inhibition (inhibition of differentiation) using mouse myoblasts. [Figure 6] 6 is a graph showing the vitronectin concentration dependency of the cell proliferation effect in the case of FIG. 5. [Figure 7] 1 is a graph showing the cell proliferation effect when chicken myoblasts are cultured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The method for producing cultured meat according to the present invention will be described below with reference to examples. Note that the following description is merely an example of one embodiment of the present invention and an example, and the present invention is not limited to the following description. The following description can be modified without departing from the spirit of the present invention.

[0011] The method for producing cultured meat according to the present invention involves culturing myoblasts in a low-serum medium containing vitronectin.

[0012] <Myoblast> The myoblasts are not particularly limited as long as they are cells other than human cells, but those taken from meat-producing vertebrates such as cows, pigs, chickens, and fish can be used. For example, "bovine myoblasts" may be bovine skeletal muscle, cardiac muscle, or smooth muscle. Skeletal muscle refers to muscle tissue in the cheeks (cheeks), temples, neck, back, chest, shoulders, waist, thighs, etc.

[0013] The term "porcine myoblasts" may refer to any of porcine skeletal muscle, cardiac muscle, and smooth muscle. Skeletal muscle is, for example, muscle tissue of the cheek (cheek), temple, neck, back, chest, shoulder, waist, and thigh.

[0014] Furthermore, the "chicken myoblast" may be any of chicken skeletal muscle, cardiac muscle, or smooth muscle. Skeletal muscle is, for example, muscle tissue of the head, neck, back, chest, shoulder, lower back, thigh, and wing (feather) regions.

[0015] The above-mentioned cells can be prepared by a method known per se. For example, in the case of myoblasts, primary myoblasts obtained by treating muscle tissue derived from a living body with a decomposing enzyme (e.g., collagenase) can be used. The primary myoblasts may be subjected to a filter treatment to remove impurities such as connective tissue. In addition, in preparing myoblasts, pluripotent stem cells such as ES cells and iPS cells, or cells induced to differentiate from somatic stem cells capable of differentiating into myoblasts can also be used.

[0016] <Vitronectin> Vitronectin is a glycoprotein present in blood and extracellular matrix, and is a so-called cell adhesion molecule. Vitronectin is obtained from animal serum, and may be human-derived vitronectin.

[0017] Vitronectin can suppress myoblast differentiation in low-serum or serum-free medium. In plate culture, vitronectin was administered at 0.14 μg / cm in low-serum medium. 2 More than 2.8μg / cm 2 Less than or equal to 0.35 μg / cm 2 More than 1.4μg / cm 2 Below 0.6 μg / cm, most preferably 2 More than 0.8μg / cm 2 It can be used below.

[0018] In three-dimensional culture, vitronectin can be utilized in low serum medium at 1.25 μg / mL to 20 μg / mL, more preferably 2.5 μg / mL to 10 μg / mL, and most preferably 4 μg / mL to 6 μg / mL.

[0019] In addition, flat plate culture is culture in a state where the cells are attached to the bottom of the culture vessel, while in 3D culture, the cells are first cultured in a high serum medium to form cell clusters, and then cultured while rotating the cells on a shaker to prevent them from attaching to the bottom of the culture dish.

[0020] <Low serum medium> A low-serum medium refers to a serum-free medium that contains 5% (weight %) or less of the total volume of serum derived from a living organism, such as fetal bovine serum (FBS) or horse serum, more preferably 2% or less. In other words, a low-serum medium contains more than 95% serum-free medium. As described above, in the present invention, a low-serum medium may contain zero serum derived from a living organism (i.e., a serum-free medium). Both DMEM and serum, which can be suitably used as serum-free medium, have a specific gravity of approximately 1 g / cm. 3 Therefore, the above ratio may be expressed as volume percent.

[0021] The medium components of the serum-free medium can be any medium that does not contain serum components derived from organisms such as cows or horses. Any known serum-free medium can be used as the serum-free medium. For example, the medium may contain Dulbecco's modified Eagle's medium (DMEM), which is a basal medium, albumin (BSA), and insulin-transferrin-selenium-ethanolamine (ITS-X).

[0022] DMEM only needs to have increased amounts of amino acids and vitamins compared to the original EME (Eagle's medium), and the contents of other ingredients may differ by about 10%. Also, the nutrient mixture F-12 Ham may not be included. However, it is not excluded that F-12 Ham may be included.

[0023] Albumin can also be used from a wide variety of sources, including egg albumin, serum albumin, milk albumin, etc. Bovine serum albumin is more preferred.

[0024] Insulin-transferrin-selenium-ethanolamine may be any commercially available product, and for example, a composition of 1000.0 mg / L insulin, 550.0 mg / L transferrin, 173.0 mg / L selenium, and 61.0 mg / L ethanolamine can be suitably used.

[0025] Furthermore, the amount of albumin relative to DMEM is preferably 3 mg / mL to 8 mg / mL, more preferably 4 mg / mL to 7 mg / mL, and most preferably 4.5 mg / mL to 5.5 mg / mL.

[0026] In addition, the concentration of ITS-X relative to DMEM is preferably 4.0 mg / mL to 7.0 mg / mL, more preferably 4.5 mg / mL to 6.5 mg / mL, and most preferably 5.0 mg / mL to 6.0 mg / mL. The above serum-free medium is called "DA-X medium".

[0027] Cholesterol can be added to the serum-free medium. Cholesterol may be of biological origin or may be synthetic, as long as it is purified. Cholesterol is preferably 5 μM to 20 μM, more preferably 10 μM to 20 μM, and most preferably 10 μM, relative to DMEM.

[0028] The low-serum medium used in the present invention may not require the addition of physiologically active substances and nutritional factors necessary for cell survival or proliferation, but may be added as necessary. These additives may be added to the medium in advance, or may be added during cell culture. The method of adding additives during culture may be in any form, such as one solution or a mixed solution of two or more kinds, and may be added continuously or intermittently.

[0029] 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, and various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.).

[0030] <Culture method> The culture method can be the same as that for ordinary cell culture. For example, the medium is premixed with the physiologically active substances and nutritional factors to be added, and is maintained at the temperature for seeding.

[0031] Myoblasts are treated with an enzyme such as collagenase to remove collagen around the cells, and after impurity removal, are seeded in a low-serum medium. Note that the myoblasts may be fixed in a serum medium before seeding in the low-serum medium.

[0032] Culture conditions were 35°C to 38°C and 3 to 6% CO. 2 The medium is changed every 1 to 2 days, and if additional components are present, they may be added at the same time as the medium is changed.

[0033] In addition, by continuing to culture separately cultured meat pieces while suppressing differentiation, it is possible to obtain thick cultured meat.

[0034] In the method for producing cultured meat according to the present invention, differentiation is inhibited by including vitronectin in the low-serum medium in which differentiation into myotubes proceeds. Therefore, in order to stop cell proliferation in myoblasts, the inhibition of differentiation can be stopped and differentiation into myotubes can be initiated by transferring the cultured cells to a low-serum medium that does not contain vitronectin.

[0035] The medium used for differentiation induction does not need to contain physiologically active substances and nutritional factors necessary for cell survival or differentiation, but may contain them as necessary. 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 (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, etc.), etc.). EXAMPLES

[0036] The effects of the present invention will be described below with reference to examples.

[0037] Mouse skeletal muscle myoblast cell line (C2C12 cells) was maintained on a single well of a 4-well plate (SPL Life Sciences, SPL-30004) in high serum medium. The medium was changed every 2 days, and C2C12 cells were dissociated with TrypLE (Gibco, 12604-013) and subcultured at a split ratio of 1:10 every 4-5 days.

[0038] For the chicken embryo myoblasts, skeletal muscle tissue was taken from the thigh of a 10-day-old chicken embryo, treated with collagenase, and filtered through a 40 μm pore filter to remove impurities. The primary myoblasts were then grown in high-serum medium overnight and then replaced with low-serum medium.

[0039] The high serum medium had the following composition: It was prepared by mixing DMEM medium (Nacalai Tesque, 08458-45) containing 10% fetal bovine serum (Gibco, 10270-106) and 1x Penicillin-Streptomycin Mixed Solution (P / S) (Nacalai Tesque, 26253-84).

[0040] Low-serum medium was prepared by mixing DMEM medium (Nacalai Tesque, 08458-45) and serum-free medium containing 1x Penicillin-Streptomycin Mixed Solution (P / S) (Nacalai Tesque, 26253-84), and adding horse serum (Sigma, H1138) at 2% (wt%) of the total medium.

[0041] Both mouse skeletal muscle myoblast cell line (C2C12 cells) and chicken embryo-derived myoblast cells have the characteristic of undergoing differentiation into myotube cells under low-serum medium conditions. Vitronectin, which has a differentiation-suppressing effect, was added together with other cell matrices at the ratios shown in the following examples. All culture conditions were 37°C, 5% CO 2 The experiment was carried out in an environment with a concentration of

[0042] <Example 1> Various cell matrices were added to a single well of a 4-well plate (SPL Life Sciences, SPL-30004) at 0.5 to 2.5 μg / cm. 2 The plate was coated with 3 × 10 mouse myoblast cells (C2C12) in 0.5 mL of DMEM supplemented with 10% fetal bovine serum. 4 The cells were seeded at a density of 100 cells / well and incubated at 37°C in 5% CO 2 After culturing for 48 hours in an environment with a concentration of 0.1% or more, the medium was replaced with DMEM containing 2% horse serum, a low-serum medium, and the cells were cultured for 72 hours.

[0043] The cell matrices were fibronectin, vitronectin, laminin, gelatin, and collagen. The control was no cell matrix. Prior to cell seeding, the extracellular matrices were precoated on a single well of a 4-well cell culture plate at the desired concentration as described below, incubated at 37°C for 1 hour, and then aspirated for use.

[0044] 2.35μg / cm 2 Fibronectin (Sigma-Aldrich, F1141) and vitronectin (FUJIFILM, 220-02041) were 1.25 μg / cm 2 , recombinant laminin 511-E8 fragment (iMatrix-511, MAX, 892011) at 0.5 μg / cm 2 The Matrigel matrix (Corning, 354234) was 2%, gelatin (Sigma, G1890) was 0.2%, and collagen type I (FUJIFILM, ASC-1-100) was 0.2%.

[0045] After culturing, the percentage of MHC (myosin heavy chain) positive cells relative to the total number of cells was examined. MHC positive cells indicate chicken myoblasts that had stopped proliferating and differentiated into myotubes.

[0046] The results are shown in Figure 1. Referring to Figure 1, the horizontal axis indicates the type of cell matrix, and the vertical axis indicates the percentage of MHC-positive cells (number of MHC-positive cells / total number of cells: unit: %). Referring to Figure 1, among the major cell matrices, vitronectin had the lowest percentage of MHC-positive cells. Furthermore, only vitronectin was able to suppress differentiation more than the control (when no cell matrix was included). This indicates that only vitronectin suppresses myoblast differentiation in low serum medium, even though this is a low serum medium that promotes rapid myoblast differentiation.

[0047] Figure 2 shows a comparison of cell numbers in the cases shown in Figure 1. Referring to Figure 2, the horizontal axis shows the sample name, and the vertical axis shows the ratio of cell numbers compared to the control cell number. By adding a cell matrix to the medium, cell increase was reduced compared to when it was not added (control). However, only when vitronectin was added did the cell number increase more than the control. This result is consistent with the result in Figure 1 that vitronectin suppresses differentiation into myotubes more than the control.

[0048] Figures 3(a) and (b) are micrographs of MHC fluorescently treated in the control and vitronectin cases. The scale bar in the lower right of the photograph is 100 μm. Comparing the control (no cell matrix) with the case where vitronectin was added, the number of bright spots was clearly reduced in the case of vitronectin, and it was also confirmed by photographic observation that differentiation of myoblasts to myotubes was suppressed. Antibody histochemical staining was used to detect MHC, a differentiation marker protein for myotubes. Myotubes were detected using a specific mouse antibody against MHC protein and a fluorescently labeled (ALEXA488) anti-mouse antibody.

[0049] Figures 3 (c) and (d) are photographs of stained cell nuclei when mouse myoblasts (C2C12) were plate-cultured in low-serum medium. The medium conditions were the same as in Figure 1. The scale bar in the lower right of the photograph is 200 μm. It can be seen that the density of bright spots is clearly denser in vitronectin compared to the control. From the above, the photographs confirm that vitronectin promotes the differentiation of myoblasts, and as a result, cell proliferation.

[0050] <Example 2> In this example, mouse myoblast cells (C2C12) and chicken myoblast cells were used. The low-serum medium was prepared by dissolving 6 × 10 cells of each type in DMEM medium containing 5 μg / mL vitronectin. 3 (C2C12) or 4×10 4 The chickens were mixed and incubated at 37°C and 5% CO 2 The cells were cultured in 3D for 5 days in a 3D atmosphere.

[0051] Figure 4 shows photographs of the results of 3D culture. The scale bars at the bottom right are all 100 μm. It can be seen that for both mouse C2C12 and chicken myoblast cells (labeled "chicken skeletal muscle"), the cell clusters were larger when vitronectin was added (Figures 4 (b) and (d)) than when they were controls (Figures 4 (a) and (c)). This means that vitronectin inhibits myoblast differentiation and efficiently promotes cell proliferation. Furthermore, 3D culture promotes cell proliferation in the cell clusters. The ability to culture in 3D is a necessary property for cultured meat.

[0052] <Example 3> In this example, the ratio of vitronectin to the medium was investigated. Mouse myoblast cells (C2C12) were seeded at different concentrations in the medium used in Example 1 and incubated at 37°C and 5% CO 2 Plate cultures were grown in the environment for 72 hours.

[0053] The results are shown in Figure 5. The horizontal axis shows samples with different vitronectin concentrations, and the vertical axis shows the number of MHC-positive cells / total nuclei (%). The vertical axis shows the number of cells (%) that differentiated from myoblasts to myotubes. Referring to Figure 5, the number of MHC-positive cells decreased in a vitronectin concentration-dependent manner.

[0054] Figure 6 shows the relative cell numbers after culturing. The horizontal axis is the vitronectin concentration as in Figure 5, and the vertical axis is the relative value with the control taken as 1.0. From these results, it can be seen that the concentration of vitronectin in the medium was 0.7 μg / cm 2 In other words, taking into account the results of Figure 5, it was found that vitronectin suppresses differentiation into myotubes in a concentration-dependent manner, but adding too much of it also suppresses cell proliferation itself.

[0055] Figure 7 shows skeletal myoblasts derived from chicken 10-day-old chick broths at a vitronectin concentration of 0.7 μg / cm 2 7 shows the results of the relative cell counts compared to the control when the same flat medium as in Example 1 was used for 6 hours of culture except that the same medium was used for 6 hours of culture. Referring to Fig. 7, the horizontal axis shows the sample type, and the vertical axis shows the cell count ratio compared to the control. Referring to Fig. 7, after 6 hours of culture, the cell count was 1.4 times that of the control. [Industrial Applicability]

[0056] The cultured meat production method of the present invention can be suitably used as a production method for artificial cultured meat.

Claims

1. A method for producing cultured meat by culturing myoblasts in a low-serum medium containing vitronectin.

2. The method for producing cultured meat described in claim 1, wherein the serum in the low serum medium is between 0% and 5% of the total volume of the low serum medium.

3. The vitronectin was added to the low serum medium at 0.14 μg / cm ２ 2.8 μg / cm or more ２ The method for producing cultured meat according to claim 1 or 2, wherein

4. The method for producing cultured meat described in claim 1 or 2, wherein the vitronectin is present in the low-serum medium at a concentration of 1.25 μg / mL or more and 20 μg / mL or less in three-dimensional culture.

5. The low serum medium is a serum-free medium, The method for producing cultured meat described in claim 1, wherein the DMEM is contained in an amount of more than 95%.

6. The method for producing cultured meat described in claim 1, further comprising a step of transferring the myoblasts to the low-serum medium that does not contain vitronectin.

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