A serum-free culture medium for the proliferation of bovine muscle stem cells and its application

Abstract

This invention relates to the fields of stem cell culture and serum-free culture media, specifically to a serum-free culture medium for the proliferation of bovine muscle stem cells and its applications. The serum-free culture medium is based on a serum-free basal culture medium, to which food-derived small molecule compounds are added. Through further combination optimization, four food-derived small molecule compounds with synergistic effects have been obtained. This serum-free culture medium can significantly promote the proliferation of bovine muscle stem cells, effectively maintain cell proliferation activity, and cells passaged using this serum-free culture medium can still maintain good differentiation potential. This provides a stable, sufficient, and safe cell source for the industrial production of cultured meat, which is beneficial for the large-scale production of bovine muscle stem cells.

Description

Technical Field

[0001] This invention relates to the fields of stem cell culture and serum-free culture media, specifically to a serum-free culture medium for the proliferation of bovine muscle stem cells and its application. Background Technology

[0002] Cultured meat, obtained by culturing animal cells in vitro, offers advantages such as environmental friendliness and sustainability. With economic growth and changing nutritional structures, cultured meat products are gradually becoming an important part of the future food supply. Muscle stem cells, capable of self-renewal and differentiation into muscle lineages, are a key cell source for the formation of muscle fibers in cultured meat. However, currently widely used traditional culture systems for cultured meat heavily rely on fetal bovine serum and exogenous growth factors. This not only leads to high culture costs but also presents significant batch-to-batch variations and risks of microbial contamination, limiting the large-scale and industrialized production of cultured meat. Therefore, developing serum-free culture systems with clearly defined components and significant effects has become a current research focus in order to construct a controllable, reproducible system suitable for food-grade production.

[0003] Currently, some progress has been made in serum-free proliferation media. International patent WO2021158103A1 first established a chemically defined serum-free culture system suitable for bovine satellite cell expansion, containing 12 components including FGF-2, IGF-1, HGF, VEGF, and PDGF-BB, achieving proliferation efficiency comparable to serum-containing systems. However, this system still relies on multiple high-cost recombinant growth factors, resulting in complex composition and limited adaptability to different cell types and large-scale application. Chinese patent CN114574433A discloses a serum-free culture medium based on DMEM / F12, supplemented with non-essential amino acids, lipids, antioxidants, recombinant growth factors, and signaling pathway modulators to support the adhesion and proliferation of myogenic cells. However, the formulation contains multiple recombinant growth factors and pathway modulators such as ROCK / p38 / Rho, which may pose cost and compliance challenges for the industrialization of cell-cultured meat for food applications.

[0004] Therefore, there is an important practical need to develop a serum-free culture medium that can efficiently promote the proliferation of bovine muscle stem cells while also being safe and scalable, so as to provide a reliable technical foundation for the industrial production of cell-cultured meat. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a serum-free culture medium containing specific food-derived small molecule compounds, thereby promoting the proliferation of bovine muscle stem cells and maintaining their differentiation potential.

[0006] Another technical problem to be solved by the present invention is to provide the application of the serum-free culture medium and a method for proliferating and culturing bovine muscle stem cells using the serum-free culture medium.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a serum-free culture medium for the proliferation of bovine muscle stem cells, the serum-free culture medium comprising a serum-free basal culture medium and a food-derived small molecule compound.

[0009] The serum-free culture medium is the "4C serum-free culture medium" described in this embodiment of the invention.

[0010] The serum-free basal medium is based on DMEM / F12, with the additional addition of L-ascorbic acid-2-phosphate trisodium salt, transferrin, sodium selenite, TGF-β3, NRG1, insulin, human recombinant albumin, bFGF, and penicillin-streptomycin solution.

[0011] In some embodiments of the present invention, the serum-free basal culture medium is based on DMEM / F12, with the following additional components: 100-200 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 10-20 μg / mL transferrin, 10-20 ng / mL sodium selenite, 0.1-0.5 ng / mL TGF-β3, 0.1-0.5 ng / mL NRG1, 10-20 μg / mL insulin, 800-1000 μg / mL human recombinant albumin, 40-60 ng / mL bFGF, and 1-3 vol% penicillin-streptomycin dual antibody solution; wherein the penicillin-streptomycin dual antibody solution contains 10000 U / mL penicillin and 10 mg / mL streptomycin.

[0012] In some embodiments of the present invention, the serum-free basal culture medium is based on DMEM / F12, with the following additional components: 200 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 20 μg / mL transferrin, 20 ng / mL sodium selenite, 0.1 ng / mL TGF-β3, 0.1 ng / mL NRG1, 20 μg / mL insulin, 800 μg / mL human recombinant albumin, 40 ng / mL bFGF, and 1 vol% penicillin-streptomycin dual antibody solution; wherein the penicillin-streptomycin dual antibody solution contains 10000 U / mL penicillin and 10 mg / mL streptomycin.

[0013] The food-derived small molecule compounds include any one or a combination of several of the following: 0.2-20 μM hesperidin, 0.2-20 μM andrographolide, 0.2-20 μM isopropyl phosphate, 0.2-20 μM aescin, 0.2-20 μM oleanolic acid, 0.2-20 μM naringin dihydrochalcone, 0.2-20 μM hesperidin, and 0.2-20 μM nomiline.

[0014] Specifically, the hesperidin, andrographolide, isopropyl phosphate, aescin, oleanolic acid, naringin dihydrochalcone, hesperidin, and nomiline were screened from 480 food-derived small molecule compounds.

[0015] In some embodiments of the present invention, the food-derived small molecule compound includes any one or a combination of several of the following: 0.2-10 μM andrographolide, 0.2-10 μM hesperidin, 0.2-10 μM hesperidin, 0.2-10 μM oleanolic acid, and 0.2-10 μM naringin dihydrochalcone.

[0016] In some embodiments of the present invention, the food-derived small molecule compounds are 0.2-10 μM andrographolide, 0.2-10 μM hesperidin, 0.2-10 μM hesperidin, 0.2-10 μM oleanolic acid and 0.2-10 μM naringin dihydrochalcone;

[0017] or,

[0018] 0.2–10 μM hesperidin, 0.2–10 μM hesperidin, 0.2–10 μM oleanolic acid and 0.2–10 μM naringin dihydrochalcone;

[0019] or,

[0020] 0.2–10 μM andrographolide, 0.2–10 μM hesperidin, 0.2–10 μM hesperidin and 0.2–10 μM naringin dihydrochalcone;

[0021] or,

[0022] 0.2–10 μM hesperidin, 0.2–10 μM hesperidin and 0.2–10 μM naringin dihydrochalcone.

[0023] In some embodiments of the present invention, the food-derived small molecule compounds are 0.2-0.5 μM andrographolide, 0.2-5 μM hesperidin, 10 μM hesperidin, 0.5-1 μM oleanolic acid and 0.5-1 μM naringin dihydrochalcone.

[0024] or,

[0025] 0.2–5 μM hesperidin, 10 μM hesperidin, 0.5–1 μM oleanolic acid and 0.5–1 μM naringin dihydrochalcone;

[0026] or,

[0027] 0.2–0.5 μM andrographolide, 0.2–5 μM hesperidin, 10 μM hesperidin and 0.5–1 μM naringin dihydrochalcone;

[0028] or,

[0029] 0.2–5 μM hesperidin, 10 μM hesperidin and 0.5–1 μM naringin dihydrochalcone.

[0030] In some embodiments of the present invention, the food-derived small molecule compounds are 0.2 μM or 0.5 μM andrographolide, 0.2 μM or 1 μM or 5 μM hesperidin, 10 μM hesperidin, 0.5 μM or 1 μM oleanolic acid and 0.5 μM or 1 μM naringin dihydrochalcone.

[0031] or,

[0032] 0.2 μM or 1 μM or 5 μM hesperidin, 10 μM hesperidin, 0.5 μM or 1 μM oleanolic acid and 0.5 μM or 1 μM naringin dihydrochalcone;

[0033] or,

[0034] 0.2 μM or 0.5 μM andrographolide, 0.2 μM or 1 μM or 5 μM hesperidin, 10 μM hesperidin and 0.5 μM or 1 μM naringin dihydrochalcone;

[0035] or,

[0036] 0.2 μM or 1 μM or 5 μM hesperidin, 10 μM hesperidin and 0.5 μM or 1 μM naringin dihydrochalcone.

[0037] In some embodiments of the present invention, the food-derived small molecule compounds are 0.5 μM andrographolide, 1 μM hesperidin, 10 μM hesperidin, 0.5 μM oleanolic acid and 0.5 μM naringin dihydrochalcone, i.e., the “5C” of the present invention.

[0038] or,

[0039] 1 μM hesperidin, 10 μM hesperidin, 0.5 μM oleanolic acid and 0.5 μM naringin dihydrochalcone, namely “5C-andrographolide” as described in this invention.

[0040] or,

[0041] 0.5 μM andrographolide, 1 μM hesperidin, 10 μM hesperidin and 0.5 μM naringin dihydrochalcone, namely the “5C-oleanolic acid” described in this invention.

[0042] or,

[0043] 1 μM hesperidin, 10 μM hesperidin and 0.5 μM naringin dihydrochalcone, namely “5C-andrographolide-oleanolic acid” as described in this invention.

[0044] Secondly, the present invention provides the application of the serum-free culture medium in the proliferation culture of bovine muscle stem cells.

[0045] The bovine muscle stem cell proliferation culture is an in vitro proliferation culture of bovine muscle stem cells.

[0046] In some embodiments of the present invention, the serum-free culture medium can significantly increase the cumulative cell number after passage to the sixth to ninth generation compared with the serum-free basal culture medium.

[0047] In some embodiments of the present invention, the serum-free culture medium can significantly increase the rate of EdU-positive cells after passage to the sixth and ninth generations of cells compared with the serum-free basal culture medium.

[0048] In some embodiments of the present invention, the serum-free culture medium can significantly improve the myotube fusion rate induced after cell passage to the sixth and ninth generations compared with the serum-free basal culture medium.

[0049] In some embodiments of the present invention, the serum-free culture medium, compared with the serum-free basal culture medium, can increase the expression of CCND1 and CCNE1 genes after the sixth generation of cell passage, and increase the expression of CCNE1 gene after the ninth generation of cell passage; at the same time, it can reduce the expression of CDKN1C and CDKN2C genes after the sixth and ninth generations of cell passage.

[0050] In some embodiments of the present invention, the serum-free culture medium can still promote the proliferation of bovine muscle stem cells within twelve passages.

[0051] Thirdly, the present invention provides a method for proliferating and culturing bovine muscle stem cells, namely, using the serum-free culture medium to proliferate and culture bovine muscle stem cells.

[0052] Fourthly, the present invention provides the application of the serum-free culture medium in the production of cell-cultured meat.

[0053] Beneficial effects:

[0054] This invention provides a serum-free culture medium that can efficiently promote the in vitro proliferation of bovine muscle stem cells while also being safe and scalable. By adding a specific combination of food-derived small molecules, selected through high-throughput screening and combination optimization, to the serum-free basal culture medium, a synergistic effect is achieved, providing a sufficient and stable cell source for the production of cultured meat. Specifically, this is reflected in the following aspects:

[0055] Firstly, regarding cell proliferation capacity and yield, compared to serum-free basal medium, the 4C serum-free medium of this invention significantly promotes the proliferation of bovine muscle stem cells. Experiments show that after passage to the sixth and ninth generations, the cell yield in the 4C serum-free medium was 1.73 and 3.63 times that of the serum-free basal medium, respectively. Furthermore, after passage to the ninth generation, the cell yield was 2.79 times that of the serum-containing proliferation medium, achieving a substantial increase in cell yield.

[0056] Secondly, the 4C serum-free medium effectively maintains the continuous proliferation activity of cells. EdU incorporation assays revealed that after six passages, the EdU-positive cell rate increased by 73.2% in the 4C serum-free medium compared to the serum-free basal medium. Even after nine passages, the 4C serum-free medium still exhibited a significantly higher EdU-positive rate compared to both serum-containing proliferation medium and serum-free basal medium, with increases of 151.96% and 99.04% respectively, demonstrating its ability to maintain cell self-renewal.

[0057] Furthermore, 4C serum-free medium maintained the differentiation potential of bovine muscle stem cells. After passage to the sixth and ninth generations, cells cultured in 4C serum-free medium successfully induced myotube formation, and compared with serum-free basal medium, the myotube fusion rate was significantly improved. Simultaneously, at the molecular level, 4C serum-free medium significantly increased the expression levels of the stem cell gene Pax7 and the cell cycle regulatory gene CCNE1, while decreasing the expression levels of CDKN1C and CDKN2C genes, revealing its role in enhancing cell yield and proliferation quality at the gene regulation level. Attached Figure Description

[0058] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0059] Figure 1 The relative absorbance values ​​of 480 food-derived small molecule compounds after culturing for 72 h.

[0060] Figure 2The relative absorbance values ​​are those of eight small molecule food-derived compounds at different concentrations after 72 h of treatment. *, **, ***, and ns indicate significance compared to serum-free basal medium, while #, ##, ###, and NS indicate significance compared to serum-containing proliferation medium.

[0061] Figure 3 The cumulative cell number after passage to the sixth generation under different concentrations of eight food-derived small molecule compounds. *, **, ***, and ns indicate significance compared to serum-free basal medium.

[0062] Figure 4 The cumulative cell number after passage to the sixth generation in each group for foodborne small molecule compounds in the single-factor deletion experiment. * and *** indicate significance compared to the serum-free basal medium group, and # and ### indicate significance compared to the 5C group.

[0063] Figure 5 This study shows the gene expression of myogenic proliferation genes Pax3, Pax7, Myf5, MyoD, and MyoG in each group after passage to the sixth generation of cells in a single-factor deletion assay using food-derived small molecule compounds. *, **, ***, and ns indicate significance markers compared to the serum-free basal medium group.

[0064] Figure 6 The cumulative number of cells passaged to the ninth generation in a multifactorial deletion experiment for foodborne small molecule compounds ( Figure 6 A in the middle), and passed down to the sixth generation ( Figure 6 B in the middle), the ninth generation ( Figure 6 Following C), a bar chart showing the cumulative cell counts in serum-containing proliferation medium (positive control group), serum-free basal medium (negative control group), and 4C serum-free medium (5C-andrographolide group, experimental group) is presented. *** and ns indicate significance compared to the serum-free basal medium group, and ### indicates significance compared to the 5C-andrographolide group.

[0065] Figure 7 Cells cultured using serum-containing proliferation medium (positive control group), serum-free basal medium (negative control group), and 4C serum-free medium (experimental group) were passaged to the sixth generation. Figure 7 A in the middle) and the ninth generation ( Figure 7 EdU staining status of B) in the image. Scale bar: 250 μm.

[0066] Figure 8 for Figure 7 Quantitative statistical analysis results of the EdU-stained positive cell rate. *** and ns indicate significance markers compared to the serum-free basal medium group.

[0067] Figure 9 Cells cultured using serum-containing proliferation medium (positive control group), serum-free proliferation medium (negative control group), and 4C serum-free medium (experimental group) were passaged to the sixth generation. Figure 9 A in the middle) and the ninth generation ( Figure 9 (B) After induction differentiation, MyHC staining results were observed 3 days later. Scale bar: 200 μm.

[0068] Figure 10 for Figure 9 Quantitative statistical analysis results of myotube fusion rate stained with MyHC immunofluorescence. *** and ns indicate significance compared to the serum-free basal culture group.

[0069] Figure 11 Cells cultured using serum-containing proliferation medium (positive control group), serum-free basal medium (negative control group), and 4C serum-free medium (experimental group) were passaged to the sixth generation. Figure 11 A in the middle) and the ninth generation ( Figure 11 Following (B), the expression of cell cycle-related genes in each group was analyzed. *, **, and *** indicate significant markers compared to the serum-free basal medium group.

[0070] Figure 12 Microscopic images of cells cultured using serum-containing proliferation medium (positive control group), serum-free basal medium (negative control group), and 4C serum-free medium (experimental group) at different passage numbers. Scale bar: 200 μm.

[0071] Figure 13 The expansion fold of cells cultured using serum-containing proliferation medium (positive control group), serum-free basal medium (negative control group), and 4C serum-free medium (experimental group) after passage to the twelfth generation. *, **, and *** indicate significance markers compared to the serum-free basal medium group. Detailed Implementation

[0072] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0073] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0074] The culture media used in the following examples are as follows:

[0075] 1. Serum-containing proliferation medium: 84 vol% DMEM / F12 cell culture medium, 15 vol% fetal bovine serum, 1 vol% penicillin-streptomycin antibiotic solution, and 5 ng / mL basic fibroblast growth factor (bFGF). The penicillin-streptomycin antibiotic solution contains 10,000 U / mL penicillin and 10 mg / mL streptomycin.

[0076] 2. Serum-free basal culture medium: 97.3 vol% DMEM / F12 cell culture medium, 200 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 20 μg / mL transferrin, 20 ng / mL sodium selenite, 0.1 ng / mL TGF-β3, 0.1 ng / mL NRG1, 20 μg / mL insulin, 800 μg / mL human recombinant albumin, 40 ng / mL bFGF, and 1 vol% penicillin-streptomycin dual antibody solution. The penicillin-streptomycin dual antibody solution contained 10000 U / mL penicillin and 10 mg / mL streptomycin.

[0077] 3. Serum-free adherent culture medium: serum-free basal culture medium without the addition of 800 μg / mL human recombinant albumin.

[0078] In the following embodiments, the rat tail collagen type I was purchased from Corning Incorporated, USA.

[0079] In the following embodiments, "vol%" refers to volume percentage.

[0080] Example 1: Screening of a library of foodborne small molecule compounds

[0081] Bovine muscle stem cells were seeded at a rate of 600 cells / well in 96-well culture dishes coated with rat tail collagen type I at a final concentration of 0.25 mg / mL. The cells were cultured in serum-containing proliferation medium at 37°C in a 5% CO2 incubator for 24 h, after which the medium was discarded. Cells were washed with PBS and then replaced with different types of culture media: (1) serum-containing proliferation medium, (2) serum-free basal medium supplemented with 480 different 10 μM dietary small molecule compounds, and (3) serum-free basal medium without the supplemented 480 different 10 μM dietary small molecule compounds. After 72 h of culture, the original culture medium was removed. CCK-8 working solution, prepared by mixing CCK-8 and DMEM / F12 medium at a volume ratio of 1:10, was added to the wells. The culture dishes were incubated in a 37°C incubator in the dark for 3–4 h. The absorbance at 450 nm was measured using a microplate reader, and the relative absorbance was calculated.

[0082] The results are as follows Figure 1As shown, 62 out of 480 foodborne small molecule compounds had relative absorbance values ​​>1. Compared with the results of culture in serum-free basal medium, 8 of these foodborne small molecule compounds (hesperidin, andrographolide, isopropyl phosphate, aescin, oleanolic acid, naringin dihydrochalcone, hesperidin, and nomiline) showed significant proliferative effects with relative absorbance values ​​>1.2 (P<0.001), while the remaining compounds did not show any proliferative effects.

[0083] Example 2: Concentration optimization of 8 candidate foodborne small molecule compounds

[0084] 1. Concentration optimization of 8 foodborne small molecule compounds

[0085] Referring to the method used in Example 1, serum-containing proliferation medium was used as the positive control group, serum-free basal medium was used as the negative control group, and serum-free basal medium was used as the experimental group with different concentrations (0.2 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) of 8 food-derived small molecule compounds added respectively. The concentrations of the 8 food-derived small molecule compounds were optimized.

[0086] The results are as follows Figure 2 As shown, the eight food-derived small molecule compounds exhibited stable proliferative effects in CCK-8, and showed no cytotoxicity and significant proliferative effects in the range of 0.2–10 μM. However, at a concentration of 20 μM, each group showed some inhibitory effect on cell viability.

[0087] 2. Passaging of bovine muscle stem cells using food-derived small molecule compounds at concentrations of 0.2–10 μM.

[0088] (1) In vitro culture of bovine muscle stem cells containing serum (positive control group)

[0089] According to 1.5×10 per hole 4Bovine muscle stem cells were seeded into 6-well culture dishes coated with a final concentration of 0.25 mg / mL rat tail collagen type I and cultured in serum-containing proliferation medium at 37°C in a 5% CO2 incubator, with the medium changed every two days. For continuous expansion, cells were passaged when they reached 60-70% confluence. The culture medium in the dishes was discarded, and each well was washed once with 2 mL PBS. After discarding the PBS, 500 μL of trypsin was added to each well and the cells were incubated at 37°C for 1-2 min. The cell status was observed under a microscope. When a large number of cells became rounded and suspended, the digestion was stopped by adding 2-3 times the amount of serum-containing proliferation medium. The bottom of the culture dish was gently agitated with a pipette to detach all the cells. The cell suspension was collected into a 15 mL centrifuge tube, centrifuged at 500 g for 5 min, and the cell pellet was collected. The cells were gently resuspended in 200 μL of serum-containing proliferation medium to obtain a cell suspension. 10 μL of the cell suspension was mixed thoroughly with 10 μL of trypan blue, and then 10 μL of the mixture was added to a cell counting chamber and counted using a cell counter. Based on the counting results, cells were seeded at an appropriate cell density in each culture dish, and the cells were evenly distributed by cross-shading. The cells were then cultured at 37°C in a 5% CO2 incubator until they reached suitable confluence before the next passage. The culture was continued until the sixth passage.

[0090] (2) In vitro culture of serum-free bovine muscle stem cells (negative control group, each experimental group)

[0091] Bovine muscle stem cells were passaged for one generation in serum-containing proliferation medium. After the bovine muscle stem cells were revived, they were counted at a ratio of 1.5 × 10⁶ cells per well. 4Resuscitated bovine muscle stem cells were transferred into culture dishes coated with rat tail collagen type I at a final concentration of 0.25 mg / mL. They were initially cultured in serum-containing proliferation medium at 37°C and 5% CO2 for 24 h to promote cell adhesion and early adaptation. After 24 h, the medium was discarded, and the cells were washed once with PBS. The medium was then replaced with serum-free basal medium (i.e., serum-free first generation) with or without 0.2–10 μM dietary small molecule compounds. Cells were cultured at 37°C and 5% CO2 until the cell confluence reached 60–70%. The culture medium was discarded, and each well was washed once with 2 mL of PBS. After discarding the PBS, each well was digested with 800 μL of TrypLE Express and incubated at 37°C for 4–5 min. Cell status was observed under a microscope. When a large number of cells became rounded and suspended, digestion was terminated by adding an equal volume of DMEM / F12 supplemented with 0.5% bovine serum albumin (BSA). Gently pipette the bottom of the culture dish to detach all the cells, collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 500 g for 5 min, collect the cell pellet, and resuspend the cells in 200 μL of serum-free adherent culture medium to obtain the cell suspension.

[0092] Mix 10 μL of cell suspension with 10 μL of trypan blue, then add 10 μL of the mixture to a cell counting chamber and count the cells using a cell counter. Based on the counting results, sort the cells at a density of 1.5 × 10⁶ cells per well. 4 1 μg / cm² of cells were evenly seeded onto a coating layer. 2 Phelanin was cultured in 6-well dishes in serum-free adherent medium with or without 0.2–10 μM dietary small molecule compound for 24 h to promote cell adhesion. After 24 h, the serum-free adherent medium was discarded, and the cells were washed once with PBS. After discarding the PBS, the culture was replaced with serum-free basal medium with or without 0.2–10 μM dietary small molecule compound and cultured until the appropriate confluence was reached for passage (i.e., serum-free second generation). Subsequent passages were performed using the same method, without further exposure to serum. Cell counts were performed at each passage, and the cells were passaged up to the sixth generation.

[0093] The sustained proliferative effect of eight food-derived small molecule compounds on cells was verified by short-term passage in the range of 0.2–10 μM. The results are as follows: Figure 3As shown, the experimental groups supplemented with 0.2 μM andrographolide, 0.5 μM andrographolide, 0.2 μM hesperidin, 1 μM hesperidin, 5 μM hesperidin, 10 μM hesperidin, 0.5 μM oleanolic acid, 1 μM oleanolic acid, 0.5 μM naringin dihydrochalcone, and 1 μM naringin dihydrochalcone showed significantly higher cumulative cell counts after six passages compared to serum-free basal medium. The experimental groups supplemented with 500 nM (0.5 μM) andrographolide, 1 μM hesperidin, 10 μM hesperidin, 500 nM (0.5 μM) oleanolic acid, and 500 nM (0.5 μM) naringin dihydrochalcone showed the most significant improvement. However, the experimental groups supplemented with aescin, nomiline, and isopropyl phosphate did not show a significant increase in cumulative cell counts during short-term passages. Based on the above results, five candidate food-derived small molecule compounds (andrographolide, hesperidin, hesperidin, oleanolic acid, and naringin dihydrochalcone) were found to significantly promote the proliferation of bovine muscle stem cells under different concentration conditions.

[0094] Example 3: Validation of the synergistic proliferative capacity of food-derived small molecule compounds and the expression of myogenic proliferative genes.

[0095] 1. Detection of the synergistic proliferative capacity of food-derived small molecule compounds (single-factor deletion experimental combination)

[0096] Using the five candidate food-derived small molecule compounds selected in Example 2 and their corresponding concentrations, the synergistic proliferative capacity of the food-derived small molecule compounds was further investigated through single-factor deletion experiments listed in Table 1. The passaging method for bovine muscle stem cells was the same as described in Example 2.

[0097] Table 1. Single-factor deletion experimental combinations of food-derived small molecule compounds in Example 3

[0098]

[0099] The results are as follows Figure 4 As shown, the cell yield in the 5C group was increased by 70.23% compared to the serum-free basal medium. Both the 5C-andrographolide and 5C-oleanolic acid groups showed more significant proliferation-promoting effects. Compared to the 5C group, the cell yield in the 5C-andrographolide group increased by 26.29%, and the cell yield in the 5C-oleanolic acid group increased by 12.88%.

[0100] 2. Detection of myogenic proliferation gene expression in bovine muscle stem cells (single-factor deletion assay combination)

[0101] Bovine muscle stem cells passaged to the sixth generation in step 1 were collected. RNA was extracted from the cells using an RNA extraction kit (Beyotime, RC102) according to the manufacturer's instructions, and the RNA concentration was measured using a Nandrop micro-spectrophotometer. cDNA was then obtained by reverse transcription using a reverse transcription kit (Beyotime, R323-01) according to the manufacturer's instructions. RT-qPCR reactions were performed using the cDNA as a template against target genes (Pax3, Pax7, Myf5, MyoD, MyoG) and the internal reference gene (GAPDH) to detect the expression of myogenic proliferation genes. The reaction was performed using Sangon Biotech 2× Universal SYBR Green qPCR Master Mix. The cDNA template obtained from reverse transcription was diluted appropriately before use. The 20 μL RT-qPCR reaction system is shown in Table 2, and the upstream and downstream primers used for the corresponding genes are shown in Table 3.

[0102] Table 2 RT-qPCR reaction system

[0103]

[0104] Table 3 Primer sequence information corresponding to myogenic proliferation genes

[0105]

[0106] result Figure 5 As shown, compared with the serum-free basal culture medium group, the 5C group and the 5C-andrographolide group significantly increased the expression levels of Pax3, MyoD and MyoG genes; it is worth noting that the 5C group, the 5C-andrographolide group and the 5C-oleanolic acid group all significantly increased the expression level of Pax7 gene.

[0107] 3. Detection of the synergistic proliferative capacity of food-derived small molecule compounds (multi-factor deletion experimental combination)

[0108] Using the five candidate foodborne small molecule compounds and their corresponding concentrations selected in Example 2, and employing the multi-factor deletion experimental combinations listed in Table 4, the study investigated whether the simultaneous removal of andrographolide and oleanolic acid could further enhance the synergistic proliferative effect. The passaging method for bovine muscle stem cells was the same as described in Example 2.

[0109] Table 4. Multifactor deletion experimental combinations of foodborne small molecules in Example 3

[0110]

[0111] The results showed that the cumulative cell number in the 5C-andrographolide group after passage to the ninth generation was significantly higher than that in the 5C-andrographolide-oleanolic acid group and the 5C-oleanolic acid group. Figure 6(A) Compared to serum-free basal medium, the 5C-andrographolide group significantly increased the cumulative cell number after six passages, with the cumulative cell number being 1.73 times that of the serum-free basal medium. Figure 6 (B in the text); the effect was more significant after passage to the ninth generation, with the cumulative cell number being 2.79 times and 3.63 times that of serum-containing proliferation medium and serum-free basal medium, respectively. Figure 6 (C). The cumulative cell counts in the 5C-oleanolic acid and 5C-andrographolide-oleanolic acid groups were significantly increased compared to the serum-free basal medium, but still significantly lower than those in the 5C-andrographolide group. Furthermore, the 5C group... Figure 4 The proliferative advantage exhibited by the culture medium significantly decreased after passage to the ninth generation, and the cumulative cell number after passage to the ninth generation was not significantly different from that of the serum-free basal medium group.

[0112] The results above show that the 5C-andrographolide group had the best effect. The 5C-andrographolide group was added to serum-free basal medium and serum-free adherent medium, respectively, and finally the preferred new serum-free basal medium (i.e., 4C serum-free medium) and serum-free adherent medium (and 4C serum-free adherent medium) for bovine muscle stem cells were obtained.

[0113] The serum-free culture medium (4C) had the following formulation: 97.3 vol% DMEM / F12 cell culture medium, 200 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 20 μg / mL transferrin, 20 ng / mL sodium selenite, 0.1 ng / mL TGF-β3, 0.1 ng / mL NRG1, 20 μg / mL insulin, 800 μg / mL human recombinant albumin, 40 ng / mL bFGF, 1 vol% penicillin-streptomycin dual antibody solution, 1 μM hesperidin, 10 μM hesperidin, 500 nM oleanolic acid, and 500 nM naringin dihydrochalcone. The penicillin-streptomycin dual antibody solution contained 10,000 U / mL penicillin and 10 mg / mL streptomycin.

[0114] The specific formula for 4C serum-free adherent culture medium is: 4C serum-free culture medium without the addition of 800 μg / mL human recombinant albumin.

[0115] Example 4: Detection of in vitro proliferation capacity of bovine muscle stem cells

[0116] Bovine muscle stem cells were passaged to the sixth and ninth generations according to the method described in Example 2. The positive control group was cultured in serum-containing proliferation medium, the negative control group was cultured in serum-free basal medium, and the experimental group was cultured in 4C serum-free medium. The in vitro proliferation capacity of bovine muscle stem cells was detected by EdU immunofluorescence assay, specifically according to the kit (Beyotime, C0078S) instructions. On the day of passage to the sixth and ninth generations, EdU (10 mM) was mixed with DMEM / F12 medium at a volume ratio of 1:500 to prepare EdU working solution (20 μM). 1 mL of old culture medium was aspirated and 1 mL of freshly prepared EdU working solution was added to make the final EdU concentration 10 μM. The cells were then incubated at 37°C and 5% CO2 for 4-5 h. After incubation, the EdU working solution was removed, the cells were washed with PBS, fixed with 4% paraformaldehyde, and then washed three times with PBS containing 3% BSA for 5 min each time. Cells were then permeated with PBS containing 0.3% Triton-X-100 for 15 min, followed by washing three times with PBS containing 3% BSA for 5 min each time. 500 μL of Click reaction solution (preparation method shown in Table 5 below) was added to each well, and the cells were incubated at room temperature in the dark for 30 min. Afterward, the cells were washed three times with PBS containing 3% BSA for 5 min each time. The nuclei were then counterstained with Hoechst 33342 for 10 min, and the cells were imaged using a fluorescence microscope. The percentage of positive cells was calculated based on the images.

[0117] Table 5 EdU Click Reaction Solution Preparation System

[0118]

[0119] The results are as follows Figure 7 and Figure 8 The EdU incorporation experiment verified the proliferation-promoting ability of the 4C serum-free medium. When passaged to the sixth generation, the EdU-positive cell rate of bovine muscle stem cells in the 4C serum-free medium group increased by 90.7% and 73.2% compared with serum-containing proliferation medium and serum-free basal medium, respectively. When passaged to the ninth generation, the EdU-positive cell rate in the 4C serum-free medium group increased by 151.96% and 99.04% compared with serum-containing proliferation medium and serum-free basal medium, respectively.

[0120] Example 5: Myotube formation ability of bovine muscle stem cells

[0121] The differentiation medium was formulated as follows: 97 vol% DMEM cell culture medium, 2 vol% horse serum, and 1 vol% penicillin-streptomycin antibiotic solution. The penicillin-streptomycin antibiotic solution contained 10,000 U / mL of penicillin and 10 mg / mL of streptomycin.

[0122] 1. In vitro differentiation of serum-containing bovine muscle stem cells: Bovine muscle stem cells obtained from sixth-generation and ninth-generation passage cultures were incubated at 3×10⁻⁶ cells / years. 5 Cells were seeded in 3.5 cm culture dishes coated with 2% Matrigel and cultured in serum-containing proliferation medium until the cells completely covered the bottom of the dish. The original medium was then removed, and the cells were washed with PBS and replaced with differentiation medium. During differentiation, the differentiation medium was changed every 48 hours after the start of induction. After 3 days of differentiation, the cells were washed with PBS and fixed with 4% paraformaldehyde.

[0123] 2. Serum-free bovine muscle stem cell in vitro differentiation: Bovine muscle stem cells obtained from sixth-generation and ninth-generation passage cultures were incubated at 1×10⁻⁶ cells / year. 5 1,000 cells were seeded on a substrate coated with 2% Matrigel and 1 μg / cm3. 2 In 3.5 cm culture dishes containing telogen efflux, cells were maintained in serum-free adherent medium / 4C serum-free adherent medium for 24 hours. After 24 hours, the medium was changed to serum-free basal medium / 4C serum-free medium. The medium was changed every two days until the cells completely covered the bottom of the culture dish. Then, the original medium was removed, and the cells were washed with PBS and replaced with differentiation medium. During differentiation, the differentiation medium was changed every 48 hours after the start of induction. After 3 days of differentiation, the cells were washed with PBS and fixed with 4% paraformaldehyde.

[0124] 3. Immunofluorescence staining: After fixing the samples overnight with 4% paraformaldehyde at 4°C, wash three times with PBS for 5 min each time. Then, permeabilize / block with 500 μL of 0.5% Triton X-100 (prepared with 1% BSA) at room temperature for 30 min, followed by washing three times with PBS for 5 min each time. Next, incubate the cells with anti-MyHC (Abcam, Cat# ab37484) overnight at 4°C, followed by washing three times with PBS for 5 min each time. Then, incubate the cells with Dylight 594 goat anti-mouse IgG (Abbkine, Cat# A23410) for 2 hours, followed by washing three times with PBS for 5 min each time. Finally, add 30 μL of DAPI to the sample wells to stain the cell nuclei. Images were taken using a fluorescence microscope.

[0125] The results are as follows Figure 9 , Figure 10As shown, compared to the serum-free basal medium group, the myotube fusion rate induced by cells in the 4C serum-free medium group increased by 45.72% after passage to the sixth generation. When the cells were passaged to the ninth generation, the myotube fusion rate induced by cells in the 4C serum-free medium group increased by 125.12% and 77.78% compared to the serum-containing proliferation medium group and the serum-free basal medium group, respectively.

[0126] Example 6: Detection of cell cycle-related gene expression in bovine muscle stem cells

[0127] Bovine muscle stem cells were passaged to the sixth and ninth generations using the method described in Example 2. The positive control group used serum-containing proliferation medium, the negative control group used serum-free basal medium, and the experimental group used 4C serum-free medium. Cells passaged to the sixth and ninth generations were collected, centrifuged, and the pellets were collected. The expression of cell cycle-related genes (CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, TP53, CDK1, CDK2, CDK4, CCND1, CCND2, CCNE1, CCNE2, E2F1) was detected using the method for detecting myogenic proliferation genes in bovine muscle stem cells described in Example 3. The upstream and downstream primers used for cell cycle-related genes are shown in Table 6.

[0128] Table 6. Primer sequence information used for cell cycle-related genes.

[0129]

[0130] The results are as follows Figure 11 As shown, serum-free 4C medium significantly increased the expression levels of CCND1 and CCNE1 genes after cell culture to the sixth generation compared to serum-free basal medium. Figure 11 (A in the text), and the expression level of the CCNE1 gene after being cultured to the ninth generation ( Figure 11 (B in the text). Furthermore, compared to serum-free basal medium, the expression levels of CDKN1C, CDKN2C, and TP53 were significantly reduced in cells cultured to the sixth generation in 4C serum-free medium (B in the text). Figure 11 In the A group, after culturing to the ninth generation, the expression levels of CDKN1C and CDKN2C were significantly reduced. Figure 11 (B in the text) This indicates that 4C serum-free medium can significantly affect the expression of cell cycle-related genes (such as CCND1 and CCNE1) and inhibit the expression of cell cycle repressors (such as CDKN1C, CDKN2C, and TP53), suggesting that 4C serum-free medium plays an important role in cell proliferation and cell cycle regulation.

[0131] Example 7: Detection of the long-term expansion capacity of bovine muscle stem cells

[0132] Bovine muscle stem cells were passaged using the passage culture method described in Example 2. The positive control group was a serum-containing proliferation medium, the negative control group was a serum-free basal medium, and the experimental group was a 4C serum-free medium. The cells were passaged a total of twelve times, and the number of cells harvested and the cell microscopic images were recorded during the passage.

[0133] The results are as follows Figure 12 Bright-field microscopy reveals that the 4C serum-free culture medium group maintained good cell morphology of bovine muscle stem cells, and the cell number was significantly higher than that of the serum-containing proliferation medium group and the serum-free basal medium group at multiple passages. Figure 13 As shown, the 4C serum-free medium significantly increased the fold increase of bovine muscle stem cells in the fourth and fifth passages compared to the serum-free basal medium. With increasing passage number, the fold increase in the 4C serum-free medium group showed a significantly higher trend compared to the serum-containing proliferation medium and the serum-free basal medium group. This indicates that the 4C serum-free medium has the potential to maintain cell proliferation during long-term culture.

[0134] This invention provides a serum-free culture medium for the proliferation of bovine muscle stem cells and its application, along with related ideas and methods. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A serum-free culture medium for the proliferation of bovine muscle stem cells, characterized in that, The serum-free culture medium was formulated as follows: 97.3 vol% DMEM / F12 cell culture medium, 200 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 20 μg / mL transferrin, 20 ng / mL sodium selenite, 0.1 ng / mL TGF-β3, 0.1 ng / mL NRG1, 20 μg / mL insulin, 800 μg / mL human recombinant albumin, 40 ng / mL bFGF, 1 vol% penicillin-streptomycin solution, 1 μM hesperidin, 10 μM hesperidin, 500 nM oleanolic acid, and 500 nM naringin dihydrochalcone. The penicillin-streptomycin bispecific antibody solution contains 10,000 U / mL of penicillin and 10 mg / mL of streptomycin.

2. The application of the serum-free culture medium according to claim 1 in the proliferation culture of bovine muscle stem cells; in, The bovine muscle stem cell proliferation culture is an in vitro proliferation culture of bovine muscle stem cells.

3. A method for proliferating and culturing bovine muscle stem cells, characterized in that, Bovine muscle stem cells were proliferated and cultured using the serum-free culture medium described in claim 1.

4. The application of the serum-free culture medium according to claim 1 in the production of bovine cell-cultured meat.

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