Food-grade natural compound combination method for promoting efficient myogenic differentiation in vitro
By adding natural compounds such as lutein, arachidic acid, and nervonic acid to the cell culture medium, the problem of low in vitro differentiation efficiency of myofibrils was solved, achieving efficient differentiation of myogenic cells and improving the quality and safety of cultured meat.
Patent Information
- Application Number
- CN202511144098.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-12-12
AI Technical Summary
In existing technologies, the in vitro differentiation efficiency of muscle fibers is low, resulting in cell-cultured meat products lacking meat flavor, nutrition, and texture. Furthermore, existing chemical inducers pose food safety risks, and there is a lack of food-grade natural compounds to improve differentiation efficiency.
Adding natural compounds such as lutein, arachidic acid, and nervonic acid to the basal differentiation induction medium, combined with horse serum and basal medium, promotes myogenic differentiation of myogenic cells.
It significantly improved the differentiation and fusion rate of myogenic cells, enhanced the in vitro myogenic differentiation capacity of muscle fibers, solved the problem of insufficient muscle fibers in cell-cultured meat, and provided high-quality animal-derived protein.
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Figure CN121109296A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for combining food-grade natural compounds to promote efficient myogenic differentiation in vitro, belonging to the field of animal cell culture and cultured meat technology. Background Technology
[0002] Cultured meat technology is an emerging future food production technology that produces meat products by artificially culturing animal cells on a large scale in vitro. Specifically, it involves extracting animal stem / adult cells from live animals, then allowing them to proliferate and differentiate in a culture medium to form the main cell types that constitute meat tissue, such as muscle fibers and fat. These cells can then be further processed using food processing techniques to create foods that mimic the texture and flavor of real meat. Compared to traditional animal husbandry, this technology no longer relies on the growth process of individual animals, achieving a shift from a farm environment to a laboratory environment. The entire production cycle of cultured meat can be shortened to 2-3 weeks, and the consumption of land and water resources can be reduced by more than 90%. However, as the in vitro culture time of muscle stem cells increases, the efficiency of in vitro differentiation of muscle fibers decreases, making it difficult to obtain sufficient myotubes and mature them into muscle fibers in a short period. This results in cultured meat products lacking myofibril proteins, which contribute to the flavor, nutrition, and texture of meat, hindering the industrialization of cultured meat.
[0003] While current research has employed gene editing and chemical inducers to improve the efficiency of myofibril differentiation in vitro, these methods may pose food safety risks and face challenges in public acceptance. Recent studies have identified a range of bioactive substances / small molecules that can promote myofibril differentiation in vitro; however, food-grade natural compounds that meet food safety requirements and can be directly used in cultured meat production are still lacking. Therefore, there is an urgent need to find a strategy to improve the efficiency of myofibril differentiation in vitro by adding safe and efficient food-grade natural compounds, which is crucial for establishing a safe and efficient myogenic differentiation technology system for cultured meat. Summary of the Invention
[0004] To address the issues of low in vitro differentiation efficiency of stem cells into myofibrils and insufficient content of key proteins such as myosin during the production of cell-cultured meat, this invention adds a natural compound that promotes myogenic differentiation of myogenic cells to the basic differentiation induction culture medium, thereby improving the differentiation efficiency of myogenic cells.
[0005] The technical solution of the present invention is as follows:
[0006] The first objective of this invention is to provide a natural compound that promotes myogenic differentiation of myocytes, said natural compound being at least one of lutein, arachidic acid, and nervonic acid.
[0007] In one embodiment of the present invention, the concentration range of lutein is 10nM-10μM, the concentration range of arachidic acid is 10nM-10μM, and the concentration range of nervonic acid is 10nM-10μM.
[0008] A second objective of the present invention is to provide a myoblast differentiation culture medium containing the myoblast differentiation inducer, horse serum, and basal culture medium.
[0009] In one embodiment of the present invention, the myogenic differentiation inducer is lutein and arachidic acid; in the culture medium, the content of lutein is 10 nM-1 μM; and the content of arachidic acid is 100 nM-1 μM.
[0010] In one embodiment of the present invention, the amount of horse serum added is 2-5%, and the basal culture medium includes basal culture media such as DMEM, DMEN / F12, and F12.
[0011] In one embodiment of the present invention, the culture medium contains 97 vol% DMEM medium, 2 vol% horse serum, 1 vol% penicillin-streptomycin solution, 1 μM lutein, and 100 nM arachidic acid.
[0012] In one embodiment of the present invention, antibiotics are added to the myoblast differentiation culture medium.
[0013] In one embodiment of the present invention, the penicillin-streptomycin solution contains 10,000 U / mL of penicillin and 10 mg / mL of streptomycin.
[0014] In one embodiment of the present invention, the myogenic stem cells include porcine muscle stem cells.
[0015] In one embodiment of the present invention, the myogenic cells include, but are not limited to, those from pigs, cattle, rabbits, rats, humans, or poultry.
[0016] A third objective of this invention is to provide a method for promoting myogenic differentiation of myogenic stem cells in vitro, wherein myogenic stem cells are contacted with the myogenic differentiation culture medium, and the myogenic stem cells include muscle stem cells.
[0017] In one embodiment of the present invention, the muscle stem cells are porcine muscle stem cells.
[0018] In one embodiment of the present invention, the contact is carried out for 3 to 4 days.
[0019] In one embodiment of the present invention, muscle stem cells are first cultured until they adhere to the culture wall and the confluence reaches 80%-90%, and then the culture medium is changed to the myoblast differentiation medium and cultured for another 3-4 days.
[0020] A fourth objective of this invention is to provide the application of the aforementioned myogenic differentiation inducer in promoting myogenic differentiation of muscle stem cells in vitro.
[0021] A fifth object of the present invention is to provide the application of the myoblast differentiation inducer, the myoblast differentiation medium, or any of the methods described herein in the field of cell culture or cell-cultured meat.
[0022] The beneficial effects of this invention are as follows:
[0023] The food-grade natural compound combination of lutein and arachidic acid provided by this invention can effectively promote the efficient differentiation of myogenic cells into myofibrils in vitro. It increases the differentiation rate of myogenic cells, which have poor progeny differentiation ability, from 23.33% to 45.18%, an increase of 21.85%; and the fusion rate from 18.83% to 33.85%, an increase of 15.02%, effectively improving the in vitro myogenic differentiation capacity of myogenic cells. This combination not only efficiently obtains various animal myofibrils from culture sources but also effectively alleviates the problem of limited synthesis of high-quality protein in cultured meat production, thereby providing humans with high-quality animal-derived protein and nutrients. Attached Figure Description
[0024] Figure 1 MyHC immunofluorescence staining images of myoblast differentiation induced by adding different concentrations of lutein in Example 1, scale bar 100 μM;
[0025] Figure 2 This is a statistical analysis graph showing the differentiation rate and fusion rate of different concentrations of lutein promoting myocardial differentiation in Example 1.
[0026] Figure 3 MyHC immunofluorescence staining images of myocardial differentiation promoted by adding different concentrations of arachidic acid in Example 2, scale bar 100 μM;
[0027] Figure 4 This is a statistical analysis chart showing the differentiation rate and fusion rate of different concentrations of arachidic acid promoting myocardial differentiation in Example 2;
[0028] Figure 5 MyHC immunofluorescence staining images of myocardial differentiation promoted by adding different concentrations of nervonic acid in Example 3, scale bar 100 μM;
[0029] Figure 6 This is a statistical analysis chart showing the differentiation rate and fusion rate of different concentrations of nervonic acid promoting myocardial differentiation in Example 3.
[0030] Figure 7 The image shows MyHC immunofluorescence staining of different combinations of natural products in Example 4 to promote myocardial differentiation. Scale bar: 100 μM.
[0031] Figure 8 This is a statistical analysis chart showing the differentiation rate and fusion rate of different combinations of natural products in Example 4 that promote myocardial differentiation.
[0032] Figure 9 The image shows MyHC immunofluorescence staining of groups I, J and E in Comparative Example 1 to promote myocardial differentiation. Scale bar: 100 μM.
[0033] Figure 10 The figure shows the statistical analysis of the differentiation rate and fusion rate of myoblasts in groups I and E in Comparative Example 1. Detailed Implementation
[0034] The reagents required for the complete culture medium and basal myogenic induction medium in the following examples, including fetal bovine serum, horse serum, and DMEM basal medium, were purchased from Thermo Fisher Scientific, with catalog numbers A5669701, 26050088, and C11995500BT, respectively. Penicillin-streptomycin solution was purchased from Nanjing Senbeiga Biotechnology Co., Ltd., with catalog number BC-CE-007. Lutein, arachidic acid, and nervonic acid were all purchased from MedChemExpress (MCE), with catalog numbers HY-N6947, HY-W004260, and HY-N2526, respectively.
[0035] Complete culture medium: 10% fetal bovine serum, 89% DMEM basal medium, and 1% penicillin-streptomycin solution; wherein the penicillin-streptomycin solution contains 10000 U / mL of penicillin and 10 mg / mL of streptomycin.
[0036] Basic myogenic induction medium (basal differentiation medium): 2% horse serum, 97% DMEM basic medium, 1% penicillin-streptomycin solution; the penicillin-streptomycin solution contains 10000 U / mL of penicillin and 10 mg / mL of streptomycin.
[0037] The P9 generation porcine muscle stem cells involved in the following examples were extracted from the muscle tissue of 5-7 day old male pigs. The extraction method was based on the literature "An efficient and economical way to obtain porcine muscletem cells for cultured meat production".
[0038] Example 1: Lutein promotes myoblast differentiation
[0039] P9 generation porcine muscle stem cells were distributed at a ratio of 1×10⁻⁶ per well. 5Cells were seeded into pre-coated Matrigel-coated clear 96-well plates. After incubation in complete culture medium for 24-48 hours, when cell confluence reached 80%-90%, different groups of cells were added to myogenic induction medium containing different concentrations of natural compounds. The myogenic induction media with different natural compounds were grouped as follows: 1) basic myogenic induction medium without natural products; 2) basic myogenic induction medium with only lutein, at concentrations of 10 nM, 100 nM, 1 μM, and 10 μM.
[0040] After adding the above-mentioned myogenic induction medium to different groups, the cells were cultured for 3-4 days at 37℃ in a 5% CO2 incubator. The medium was changed daily, and the changes in cell morphology during differentiation were observed daily under an inverted microscope. After differentiation, the effect of myogenic differentiation was identified by immunofluorescence.
[0041] The immunofluorescence assay steps are as follows: First, aspirate the culture medium from the wells and wash the remaining differentiation medium with PBS. After removing the PBS, fix the cells by incubating with 4% paraformaldehyde (w / v) at room temperature in the dark for 15 min. Remove the paraformaldehyde and wash the wells with PBS to remove any remaining paraformaldehyde. Add 0.5% Triton X-100 and permeate at room temperature for 15 min. Remove the solution and wash once with PBS. Then add blocking buffer and block at room temperature for 30 min. Remove the solution and wash once with PBS. After fixation, permeabilization, and blocking, incubate with antibody: add MYH3 antibody (1:500) to each well. Incubate overnight at 4°C. After removing the primary antibody, wash three times with PBS. Add a primary antibody of the same genus as the primary antibody. Cells were incubated with secondary antibody Plus 488 (1:500) at 37°C in the dark for 2 hours. After removing the secondary antibody, the cells were washed three times with PBS. Finally, PBS solution was added to soak the cells, and fluorescence images were captured under an inverted fluorescence microscope. ImageJ software was used to analyze and calculate the data from the images. Myoblastic differentiation rate: the proportion of MYH3-positive cell nuclei to the total number of cell nuclei; Myoblastic fusion rate: the proportion of nuclei of fused myofibrils (MYH3-positive cells with ≥5 nuclei) to the total number of cell nuclei.
[0042] The results are as follows Figure 1 As shown in the figure, the groups with different concentrations of lutein added to the myoblast differentiation medium exhibited better cell differentiation, with more and longer myotubes visible in the field of view. Based on this, the differentiation rates of different groups were calculated, and the results are as follows: Figure 2As shown, the addition of 1 μM lutein had the best effect on promoting differentiation. After treatment with 1 μM lutein, the cell differentiation rate was 36.78%, which was 1.25 times higher than that induced by the basal differentiation medium (29.33%). Regarding the fusion rate, the cell fusion rate after treatment with 1 μM lutein was 27.10%, which was 1.38 times higher than that induced by the basal differentiation medium (19.66%). These results indicate that the addition of lutein to the basal differentiation medium promoted myocardial differentiation more effectively than the differentiation induced by the basal differentiation medium itself.
[0043] Example 2: Arachidonic acid promotes myoblast differentiation
[0044] Porcine muscle stem cells were distributed at a ratio of 1×10⁻⁶ per well. 5 Cells were seeded into pre-coated Matrigel-coated clear 96-well plates. After incubation in complete culture medium for 24-48 hours, when cell confluence reached 80%-90%, different groups of cells were added to myoblastic induction medium containing different concentrations of natural compounds. The myoblastic induction mediums with different natural compounds were grouped as follows: 1) basic myoblastic induction medium without natural products; 2) basic myoblastic induction medium with only arachidic acid added, at concentrations of 10 nM, 100 nM, 1 μM, and 10 μM.
[0045] After adding the above-mentioned myogenic induction medium to different groups, the cells were cultured for 3 days at 37°C in a 5% CO2 incubator. The medium was changed daily, and the changes in cell morphology during differentiation were observed daily under an inverted microscope. After differentiation, the effect of myogenic differentiation was identified by immunofluorescence.
[0046] The results are as follows Figure 3 As shown in the figure, the myoblast differentiation media with different concentrations of arachidonic acid exhibited better cell differentiation, with more and longer myotubes visible in the field of view. Based on this, the differentiation rates of different groups were calculated, and the results are as follows: Figure 4 As shown, the addition of 100 nM arachidic acid (RAA) demonstrated the best differentiation-promoting effect. After treatment with 100 nM RAA, the cell differentiation rate was 41.41%, which was 1.41 times higher than that induced by the basal differentiation medium (29.33%). Regarding fusion rate, the cell fusion rate after treatment with 100 nM RAA was 32.59%, which was 1.65 times higher than that induced by the basal differentiation medium (19.66%). These results indicate that the addition of RAA to the basal differentiation medium significantly promoted myocardial differentiation compared to the basal differentiation medium-induced differentiation.
[0047] Example 3: Nervonic acid promotes myoblastic differentiation
[0048] Porcine muscle stem cells were distributed at a ratio of 1×10⁻⁶ per well. 5Cells were seeded into pre-coated Matrigel-coated clear 96-well plates. After incubation for 24-48 hours with complete culture medium, when cell confluence reached 80%-90%, different groups of cells were added to myogenic induction medium containing different concentrations of natural compounds. The myogenic induction mediums with different natural compounds were grouped as follows: 1) basic myogenic induction medium without natural products; 2) basic myogenic induction medium with only nervonic acid added, at concentrations of 10 nM, 100 nM, 1 μM, and 10 μM.
[0049] After adding the above-mentioned myogenic induction medium to different groups, the cells were cultured for 3 days at 37°C in a 5% CO2 incubator. The medium was changed daily, and the changes in cell morphology during differentiation were observed daily under an inverted microscope. After differentiation, the effect of myogenic differentiation was identified by immunofluorescence.
[0050] The results are as follows Figure 5 As shown in the figure, the groups with different concentrations of nervonic acid added to the myoblast differentiation medium exhibited better cell differentiation, with more and longer myotubes visible in the field of view. Based on this, the differentiation rates of different groups were calculated, and the results are as follows: Figure 6 As shown, the addition of 100 nM nervonic acid had the best effect on promoting differentiation. After treatment with 100 nM nervonic acid, the cell differentiation rate was 35.51%, which was 1.21 times higher than that induced by the basal differentiation medium (29.33%). Regarding the fusion rate, the cell fusion rate after treatment with 100 nM nervonic acid was 26.75%, which was 1.36 times higher than that induced by the basal differentiation medium (19.66%). These results indicate that the addition of nervonic acid to the basal differentiation medium promoted myocardial differentiation more effectively than the differentiation induced by the basal differentiation medium itself.
[0051] Example 4: Combination of natural products to promote myoblastic differentiation
[0052] Based on the optimal concentrations of added natural products described in Examples 1, 2, and 3, three natural compounds were combined and added to the basal myoblastic induction medium, and compared with basal myoblastic induction medium containing only one natural compound. The groups are as follows:
[0053] Group A: Basic myogenic induction culture medium without the addition of natural compounds;
[0054] Group B: Basic myogenic induction medium supplemented only with lutein at a concentration of 1 μM;
[0055] Group C: Basic myogenic induction medium supplemented only with arachidic acid at a concentration of 100 nM;
[0056] Group D: Basic myogenic induction medium supplemented only with nervonic acid, with a nervonic acid concentration of 100 nM;
[0057] Group E: Basic myogenic induction medium supplemented with lutein and arachidic acid, with lutein concentration of 1 μM and arachidic acid concentration of 100 nM;
[0058] Group F: Basic myogenic induction medium supplemented with lutein and nervonic acid, with lutein concentration of 1 μM and nervonic acid concentration of 100 nM;
[0059] Group G: Basic myogenic induction medium supplemented with arachidic acid and nervonic acid, with arachidic acid concentration of 100 nM and nervonic acid concentration of 100 nM;
[0060] Group H: Basic myogenic induction medium supplemented with lutein, arachidic acid and nervonic acid. The concentration of lutein was 1 μM, the concentration of arachidic acid was 100 nM and the concentration of nervonic acid was 100 nM.
[0061] After adding the above-mentioned myogenic induction medium to different groups, the cells were cultured for 3 days at 37°C in a 5% CO2 incubator. The medium was changed daily, and the changes in cell morphology during differentiation were observed daily under an inverted microscope. After differentiation, the effect of myogenic differentiation was identified by immunofluorescence.
[0062] The results are as follows Figure 7 As shown, compared to single-compound groups B, C, and D, the combinations of three natural products E, F, and G achieved better myogenic effects. The differentiation and fusion rates of groups A and H were compared, and the results are as follows: Figure 8 As shown, the differentiation rate of group E reached 45.18%, which was more than 5.78% higher than that of the single compound treatment group (lutein 34.64%, arachidic acid 39.40%, nervonic acid 34.05%), and 21.85% higher than that of group A (23.33%) without the addition of natural compounds. Its myoblast fusion rate (≥5-nucleus myofibrils) reached 33.85%, which was more than 5.60% higher than that of the single compound treatment group (lutein 25.27%, arachidic acid 28.25%, nervonic acid 24.75%), and 15.02% higher than that of group A (18.83%) without the addition of natural compounds. These results indicate that the addition of the combination of lutein and arachidic acid to the basal myoblast induction medium resulted in the best myoblast-promoting effect.
[0063] Comparative Example 1
[0064] Arachidonic acid was added to the basic myogenic induction medium to induce differentiation, designated as Group I. Arachidonic acid was replaced with arachidonic acid in Group E of Example 4 for induction differentiation, designated as Group J. The concentration of arachidonic acid used was 100 nM. Cells were cultured for 3 days at 37°C in a 5% CO2 incubator, with the medium changed daily. Cell morphology changes during differentiation were observed daily under an inverted microscope. After differentiation, immunofluorescence was used to assess the effectiveness of myogenic differentiation.
[0065] like Figure 9 As shown in the figure, compared with group E, the myogenic effect of group I (adding arachidonic acid alone) and group J (adding arachidonic acid and lutein) was not significant. The differentiation rate and melting rate of groups I and J were calculated, as follows: Figure 10 As shown, the differentiation rate (32.48%) and fusion rate (24.83%) of group I were significantly lower than those of group E (45.18%) and fusion rate (33.85%). The differentiation rate and fusion rate of group J, which replaced arachidonic acid with arachidonic acid, were significantly lower than those of group E. This indicates that the myogenic effect of arachidonic acid is significantly lower than that of the combination of lutein and arachidonic acid. The myogenic effect of the combination of arachidonic acid and lutein is also lower than that of the combination of lutein and arachidonic acid. This further demonstrates that the addition of lutein and arachidonic acid to the basic myogenic induction medium has the best myogenic effect.
[0066] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A myogenic differentiation inducer, characterized in that, The inducer is at least one of lutein, arachidic acid, and nervonic acid; the concentration of lutein used is 10 nM-10 μM, the concentration of arachidic acid used is 10 nM-10 μM, and the concentration of nervonic acid used is 10 nM-10 μM.
2. A myogenic differentiation culture medium, characterized in that, The myogenic differentiation medium contains the myogenic differentiation inducer of claim 1, horse serum, and basal medium.
3. The myoblast differentiation culture medium as described in claim 2, characterized in that, The myogenic differentiation inducers are lutein and arachidic acid; the myogenic differentiation culture medium contains 10 nM-1 μM of lutein and 100 nM-1 μM of arachidic acid.
4. The myoblast differentiation culture medium as described in claim 3, characterized in that, The amount of horse serum added is 2-5%, and the basal culture medium includes DMEM, DMEN / F12 or F12.
5. The myoblast differentiation culture medium according to any one of claims 2 to 4, characterized in that, Antibiotics were added to the myogenic differentiation culture medium.
6. A method for promoting myogenic differentiation of myogenic stem cells in vitro, characterized in that, Myogenic stem cells are contacted with the myogenic differentiation culture medium according to any one of claims 2 to 5, wherein the myogenic stem cells include muscle stem cells.
7. The method as described in claim 6, characterized in that, The contact lasts for 3 to 4 days.
8. The method as described in claim 7, characterized in that, First, culture the muscle stem cells until they adhere to the culture vessel and reach a confluence of 80%-90%. Then, change the culture medium to the aforementioned myogenic differentiation medium and continue culturing for 3-4 days.
9. The use of the myogenic differentiation inducer of claim 1 in promoting myogenic differentiation of muscle stem cells in vitro.
10. The use of the myogenic differentiation inducer of claim 1, or the myogenic differentiation culture medium of any one of claims 2 to 5, or the method of any one of claims 6 to 8, in the field of cell culture or cell-cultured meat.