Muscle stem cell isolation, extraction and novel hydrolysate culture system and application thereof

Abstract

The application belongs to the field of cell culture, and discloses a bovine muscle stem cell in-vitro separation and culture method, a culture medium and application thereof. The muscle stem cell in-vitro culture method of the application is to culture muscle stem cells by using a cell culture medium of non-animal source hydrolysate. The application further discloses a culture medium for culturing the muscle stem cells. By using the culture medium of the application, muscle stem cells with ideal cell quantity and cell morphology can be obtained on the basis of no or less serum addition, and the muscle stem cells can still maintain stemness and differentiation potential after continuous passage for multiple times. The application utilizes the in-vitro cultured muscle stem cells for the field of cell culture meat, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and particularly to the field of cell culture related to muscle stem cells, wherein the cultured muscle stem cells can be used for the research and production of cultured meat. Background Technology

[0002] Globally, with the growth of the world's population and the further improvement of the economic status of developing countries, the demand for meat and other animal products will continue to increase. Relying on traditional, animal-based production methods is inefficient and risky in meeting this growing demand. Animal farming, especially large-scale, intensive animal husbandry, is a major contributor to environmental stress and raises concerns about sustainable food security, worker safety, public health, and animal ethics. In recent years, the cell-cultured meat industry has developed rapidly. Cell-cultured meat has the potential to provide a significant amount of animal protein, contributing to improved global food security while also benefiting human health, the environment, and animal welfare.

[0003] The seed cells for cultured meat are generally muscle stem cells. Muscle stem cells are a type of stem cell located below the basement membrane of muscle tissue. They are present in very small numbers and are generally in a quiescent state. Under certain conditions (such as muscle tissue damage), muscle stem cells are activated and can differentiate into muscle cells. Furthermore, muscle cells can fuse together to form multinucleated cells, i.e., myofibrils. In the cultured meat industry, the primary and most critical technical challenge lies in successfully isolating and purifying muscle stem cells. Secondly, the primary cells obtained also face problems such as short culture cycles and easy cell aging and death.

[0004] Existing methods for isolating muscle stem cells are all for relatively small animals such as pigs, rabbits, and mice. There are almost no systematic methods for isolating, culturing, and identifying muscle stem cells from large animals like cattle. Therefore, it is crucial to develop a method for isolating and culturing bovine muscle stem cells that can be applied to cell culture meat research.

[0005] In recent years, to reduce the cost of cell culture (mainly the cost of serum and growth factors), hydrolysates have been widely used in the large-scale culture of some cell types (such as CHO, 293, etc.). In the cultured meat industry, there are few reports of using purely non-animal-derived hydrolysates in the basal culture medium for the culture and expansion of muscle stem cells, in order to reduce the cost of serum used in the large-scale expansion of cells during meat culture. Meanwhile, to maintain the proliferation of primary muscle stem cells, growth factor FGF2 is widely used in cell culture. However, the high cost of growth factors is a significant factor hindering further large-scale cell culture. Therefore, developing a hydrolysate and low-cost growth factor culture protocol suitable for muscle stem cells is crucial. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for isolating and culturing primary muscle stem cells, a culture medium and its application. This invention is particularly applicable to the culture of bovine primary muscle stem cells, and the obtained bovine muscle stem cells can be further applied to the research of cultured meat, which can fill the technical gaps in bovine muscle stem cell isolation methods and culture methods.

[0007] The first aspect of the present invention provides the use of a non-animal-derived hydrolysate as a serum substitute in the culture of muscle stem cells.

[0008] A second aspect of the present invention provides a muscle stem cell culture medium, the muscle stem cell culture medium comprising a basal culture medium, a non-animal hydrolysate, and FGF2.

[0009] A third aspect of the present invention provides the use of the above-described muscle stem cell culture medium in the in vitro culture of muscle stem cells.

[0010] The fourth aspect of the present invention provides a method for in vitro culture of muscle stem cells, wherein muscle stem cells are cultured in vitro using the muscle stem cell culture medium described above.

[0011] The fifth aspect of the present invention provides a muscle stem cell prepared by the in vitro culture method of muscle stem cells as described above.

[0012] The sixth aspect of the present invention provides a cultured meat, wherein the cultured meat is obtained by culturing muscle stem cells as described above under suitable conditions for forming muscle fibers and texture.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] If the culture method or culture medium cannot meet the requirements for long-term in vitro culture and multiple passages of muscle stem cells, obtaining a sufficient number of muscle stem cells would require very large muscle tissue samples for isolation and purification, which is practically infeasible. The non-animal-derived hydrolysate described in this invention can promote the proliferation of muscle stem cells in vitro and maintain their stemness. Using the in vitro culture and passage method for muscle stem cells of this invention allows for the large-scale expansion of muscle stem cells in vitro while maintaining their stemness, thereby significantly increasing the number of stem cells available for meat cell culture. In the culture medium of this invention, muscle stem cells can proliferate multiple times (four or more generations) and continue to maintain their stemness and differentiation potential after continuous passage.

[0015] This invention employs a method that combines non-animal-derived hydrolysates, especially yeast hydrolysates, with FGF2. On the one hand, this greatly reduces the amount of serum used in the culture of muscle stem cells (preferably bovine muscle stem cells). On the other hand, under these culture conditions, the cell passages can be performed at a level comparable to those achieved with the combination of normal serum and FGF2.

[0016] In the culture medium described in this invention, both domestically produced and imported FGF2 can effectively promote the proliferation of muscle stem cells while maintaining their stemness. Therefore, domestically produced FGF2 can be used to replace imported FGF2, significantly reducing the cost of culturing muscle stem cells.

[0017] This invention provides a complete protocol for cell isolation, culture, and identification, offering theoretical and practical guidance for obtaining seed cells for cultured meat. The hydrolysate selection process involved testing and elucidation of its components, and the use of the hydrolysate provides valuable insights for subsequent culture expansion protocols. This invention not only solves the problem of obtaining seed cells for cultured meat but also offers significant guidance for addressing the serum cost issue in meat culture. Cultured meat can, to some extent, address environmental, animal welfare, and meat supply shortages, and this invention provides strong technical support for this. Attached Figure Description

[0018] Figure 1 These are photomicrographs of bovine muscle stem cells isolated and cultured for 1 to 7 days, as shown in Example 1.

[0019] Figure 2 Identification of PAX7, a stem protein in bovine muscle stem cells, for Example 1.

[0020] Figure 3 Flow cytometry identification of CD29 / CD56, the stem protein on the surface of bovine muscle stem cells, for Example 1.

[0021] Figure 4 Example 1 compares the effects of domestically produced FGF2 and imported FGF2 on muscle stem cell culture.

[0022] Figure 5 The images are micrographs of fourth-generation (P1-P4) bovine muscle stem cells cultured using different hydrolysates and domestic FGF2 in Example 1. Detailed Implementation

[0023] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] This invention reveals that bovine muscle stem cells, when cultured in a medium with an appropriate amount of non-animal-derived hydrolysate, can proliferate well even with very low serum concentrations. In this novel muscle stem cell culture medium, bovine muscle stem cells can be passaged multiple times, and the number and morphology of the obtained cells are similar to those obtained in high-serum culture media. This indicates that the novel muscle stem cell culture medium of this invention can replace existing high-serum culture media, significantly reducing the cost of cell culture. More importantly, these cells cultured in this novel muscle stem cell culture medium with an appropriate amino acid content can be used in the field of cultured meat, greatly reducing the cost of culturing bovine muscle cells.

[0025] This invention is the first to propose the use of non-animal-derived hydrolysates as serum substitutes in the culture of muscle stem cells. Using these non-animal-derived hydrolysates in the culture of muscle stem cells can completely or at least partially replace serum, significantly reducing the amount of serum required.

[0026] The non-animal-derived hydrolysate is preferably a yeast hydrolysate. The yeast hydrolysate can be any suitable yeast hydrolysate. In some preferred embodiments, the yeast hydrolysate is a baker's yeast (Saccharomyces cerevisiae) hydrolysate; the baker's yeast hydrolysate can be obtained, for example, by protease hydrolysis; the protease includes, but is not limited to, one or more of papain, bromelain, pepsin, trypsin, Aspergillus niger acidic protease, flavor protease, Bacillus subtilis protease, peptidase, alkaline protease, and neutral protease. In some preferred embodiments, the yeast hydrolysate contains crude protein, amino acids, small peptides, and nucleic acids, and the mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 25-50; the mass percentages of polypeptides with molecular weights greater than 2 kDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, and less than 180 Da in the total polypeptides are 0.5-0.8%, 0.8-15%, 15-38%, 40-60%, and 10-15%, respectively. In some preferred embodiments, the mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 46; the mass percentages of polypeptides with molecular weights greater than 2 kDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, and less than 180 Da in the total polypeptides are 0.6%, 1.14%, 20.06%, 56.55%, and 21.65%, respectively. Products that meet these requirements are commercially available products, such as the commercially available product in Table 1, namely yeast hydrolysate #1 (Angel, product number CM-YE-01). In some other preferred embodiments, the mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 30.8; the mass percentages of polypeptides with molecular weights greater than 2 kDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, and less than 180 Da in the total polypeptides are 0.73%, 10.24%, 33.35%, 42.02%, and 13.66%, respectively. Products that meet these requirements are commercially available products, such as the commercially available product in Table 1, namely yeast hydrolysate #2 (Angel, product number CM-YE-03).

[0027] In some preferred embodiments, the method for preparing the baker's yeast hydrolysate includes: using baker's yeast (Saccharomyces cerevisiae) as raw material and obtaining it through a bio-directed degradation method; specifically, using baker's yeast cultured with high-density fermentation regulation technology as raw material, and employing bio-directed degradation and membrane filtration technology, followed by concentration and spray drying to obtain powdered yeast hydrolysate; the yeast hydrolysate contains crude protein, amino acids, small peptides, and nucleic acids, and the mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 25-50; the mass percentages of peptides with molecular weights greater than 2 kDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, and less than 180 Da in the total peptides are 0.5-0.8%, 0.8-15%, 15-38%, 40-60%, and 10-15%, respectively. In some preferred embodiments, the mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 46; the mass percentages of polypeptides with molecular weights greater than 2 kDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, and less than 180 Da in the total polypeptides are 0.6%, 1.14%, 20.06%, 56.55%, and 21.65%, respectively. Products that meet these requirements are commercially available products, such as the commercially available product in Table 1, namely yeast hydrolysate #1 (Angel, product number CM-YE-01). In some other preferred embodiments, the mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 30.8; the mass percentages of polypeptides with molecular weights greater than 2 kDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, and less than 180 Da in the total polypeptides are 0.73%, 10.24%, 33.35%, 42.02%, and 13.66%, respectively. Products that meet these requirements are commercially available products, such as the commercially available product in Table 1, namely yeast hydrolysate #2 (Angel, product number CM-YE-03).

[0028] The present invention also provides a novel bovine muscle stem cell culture medium, which is a cell culture medium supplemented with an appropriate amount of non-animal hydrolysate, which can be prepared by adding an appropriate amount of non-animal hydrolysate to a basal cell culture medium; comprising basal culture medium, non-animal hydrolysate and FGF2.

[0029] The novel muscle stem cell culture medium described in this invention is applicable to any suitable animal muscle stem cells. Preferably, it is bovine muscle stem cells, porcine muscle stem cells, rabbit muscle stem cells, etc.; more preferably, it is bovine muscle stem cells.

[0030] In the novel muscle stem cell culture medium of this invention, the non-animal-derived hydrolysate is yeast hydrolysate. The preparation method of the yeast hydrolysate includes: using yeast as raw material, and obtaining it through a bio-directed degradation method. Specifically, the preparation method of the yeast hydrolysate includes: using baker's yeast cultured with high-density fermentation regulation technology as raw material, employing bio-directed degradation and membrane filtration technology, and obtaining powdered yeast hydrolysate through concentration and spray drying. The mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 25-50; and / or, the mass percentages of polypeptides with molecular weights greater than 2 kDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, and less than 180 Da in the total polypeptides are 0.5-0.8%, 0.8-15%, 15-38%, 40-60%, and 10-15%, respectively. In some preferred embodiments, the yeast hydrolysate is yeast hydrolysate #1 and yeast hydrolysate #2 in Table 1.

[0031] In the novel muscle stem cell culture medium of the present invention, the concentration of the non-animal-derived hydrolysate in the muscle stem cell culture medium is 0.1-20 mg / mL, and can be 0.1-0.5, 0.5-1, 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, 9.5-10, 10-11, 11-12, 12-13, 13-14, 14-16, 16-18, or 18-20 mg / mL. Preferably, the concentration of the non-animal-derived hydrolysate in the muscle stem cell culture medium is 3-8 mg / mL; more preferably, it is 4-6 mg / mL. More preferably, it is 1 mg / mL.

[0032] The novel muscle stem cell culture medium of the present invention may also contain FGF2. The concentration of FGF2 in the muscle stem cell culture medium is 1-50 ng / ml, and can be 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, 9.5-10, 10-11, 11-12, 12-13, 13-14, 14-16, 16-18, 18-20, 20-21, 21-22, 22-23, 23-24, 24-26, 26-28, 28-30, 30-35, 35-40, 40-45, or 45-50 ng / ml. Preferably, the concentration of the non-animal-derived hydrolysate in the muscle stem cell culture medium is 5-20 ng / ml; more preferably, it is 8-12 ng / ml mg / L. More preferably, it is 10 ng / ml.

[0033] In the novel muscle stem cell culture medium of this invention, in addition to the basal culture medium components and the addition of an appropriate amount of non-animal-derived hydrolysate, serum may or may not be included to save costs. When serum is included, its content is very low. In some embodiments, the volume of serum accounts for 0.5-2% of the volume of the basal culture medium, which can be 0.5-1%, 1-1.5%, or 1.5-2%; preferably, the serum added in this invention is 0.5%.

[0034] The novel muscle stem cell culture medium of this invention generally comprises the following components: balanced saline, pH adjusting solution, antibiotics, animal serum, vitamins, glucose, etc. The balanced saline may contain calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium fluoride, sodium chloride, sodium phosphate, etc.; the pH adjusting solution may contain sodium bicarbonate, HEPES solution, sodium pyruvate, etc.; the antibiotics may contain penicillin, streptomycin, etc.; commonly used animal serums include bovine serum and horse serum. Vitamins may contain choline chloride, folic acid, inositol, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, vitamin B6, riboflavin, thiamine, etc.

[0035] In some embodiments, the basal culture medium of the novel muscle stem cell culture medium of the present invention may be selected from at least one of DMEM, RPMI 1640, MEM, DEME / F12, F10, CD293, medium 231, medium 106, and basal culture media modified therefrom. Specifically, embodiments of the present invention preferably use a bovine muscle stem cell culture medium in which various non-animal hydrolysates are added to DMEM medium.

[0036] In some preferred embodiments, the novel muscle stem cell culture medium is a culture medium containing non-animal hydrolysate, including: DMEM medium, non-animal hydrolysate, FGF2, and serum.

[0037] The concentration of the non-animal-derived hydrolysate in the muscle stem cell culture medium is 0.1-20 mg / mL, and can be 0.1-0.5, 0.5-1, 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, 9.5-10, 10-11, 11-12, 12-13, 13-14, 14-16, 16-18, or 18-20 mg / mL. Preferably, the concentration of the non-animal-derived hydrolysate in the muscle stem cell culture medium is 3-8 mg / mL; more preferably, it is 4-6 mg / mL. More preferably, it is 1 mg / mL.

[0038] The concentration of FGF2 in the muscle stem cell culture medium is 1-50 ng / ml, and can be 1-1.5, 1.5-2, 2-2.5, 2.5-3, 3-3.5, 3.5-4, 4-4.5, 4.5-5, 5-5.5, 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7.5-8, 8-8.5, 8.5-9, 9-9.5, 9.5-10, 10-11, 11-12, 12-13, 13-14, 14-16, 16-18, 18-20, 20-21, 21-22, 22-23, 23-24, 24-26, 26-28, 28-30, 30-35, 35-40, 40-45, or 45-50 ng / ml. Preferably, the concentration of the non-animal-derived hydrolysate in the muscle stem cell culture medium is 5-20 ng / ml; more preferably, it is 8-12 ng / ml. More preferably, it is 10 ng / ml.

[0039] The serum volume accounts for 0.5-2% of the muscle stem cell culture medium volume, which can be 0.5-1, 1-1.5, or 1.5-2%; preferably, the serum added in this invention is 1%.

[0040] In some embodiments, the preparation steps of the culture medium containing non-animal hydrolysate include: first weighing the appropriate amount of hydrolysate, dissolving it in DMEM culture medium, and then adding serum and FGF2 to prepare a culture medium containing non-animal hydrolysate.

[0041] In some preferred embodiments, the present invention provides a method for culturing bovine muscle stem cells, comprising the steps of:

[0042] (1) Organize collection

[0043] Collect bovine muscle tissue and perform preliminary sterilization treatment.

[0044] (2) Tissue fragmentation and digestion

[0045] The beef muscle tissue was broken down and then digested with protease to obtain a digestive juice.

[0046] In step (1), the bovine muscle tissue can be any suitable muscle tissue; since frozen tissue will cause cell death and cannot be extracted after a long time, frozen muscle tissue is not recommended; the present invention prefers fresh tissue; more preferably, fresh bovine muscle tissue with a slaughter time of less than 3 hours.

[0047] Step (1) also includes the step of removing surface tissue before initial sterilization.

[0048] In step (1), the preliminary sterilization treatment can be performed using any suitable sterilizing solution. Preferably, 75% alcohol is used for sterilization, more preferably, alcohol in an ice bath is used. The sterilization time can be set according to actual needs, and can be 1-10 minutes, preferably 2 minutes.

[0049] In step (1), the preliminarily sterilized tissue is transferred to a PBS buffer containing penicillin-streptomycin antibiotics. Preferably, a PBS buffer containing penicillin-streptomycin antibiotics is used in an ice bath. The concentration of the penicillin-streptomycin antibiotics can be set according to actual needs; in this invention, the concentration of penicillin-streptomycin antibiotics in the PBS buffer is preferably 10% (v / v). The purpose of adding the antibiotics is to remove bacteria on the tissue and avoid bacterial contamination during subsequent tissue extraction and cell culture.

[0050] In step (2), the disruption is performed in a sterile environment. Care should be taken to avoid mechanical shearing during disruption to prevent cell damage. Suitable tools, such as surgical scissors, can be used to disrupt the cells. The tissue can be disrupted to any size, but it should be digestible without damaging the cells. In some preferred embodiments, the tissue is disrupted to 0.5 mm. 3 about.

[0051] In step (2), the protease is selected from trypsin, type I collagenase, and type III collagenase; preferably, it is trypsin.

[0052] In step (2), the digestion temperature can be selected according to actual needs, such as 30-40℃, for example 30-32℃, 32-34℃, 34-36℃, 36-38℃, 38-40℃, preferably 32-38℃; in a more preferred embodiment, the digestion temperature is 37℃.

[0053] In step (2), the digestion time can be selected according to actual needs. For example, it can be determined by the form of the digested tissue fluid, etc., and can be 0.5-5 hours, such as 0.5, 1, 2, 3, 4, 5 hours; in a more preferred embodiment, the digestion time is 1 hour.

[0054] In step (2), during the digestion process, in order to ensure that the tissue can be fully digested, it is preferable to shake the tissue fluid at regular intervals, and more preferably to shake it every 5 minutes.

[0055] In step (2), the digestion is terminated by adding DMEM medium, preferably by adding DMEM medium containing 20% ​​(v / v) serum.

[0056] (3) Tissue filtration

[0057] After digestion is complete, the digestive fluid is filtered, and then the filtrate is centrifuged to obtain cell precipitate.

[0058] In step (3), the filtration is performed using a 40-300μm mesh sieve; preferably, the filtration is first performed by coarse sieving through a large-pore mesh sieve, and then by secondary filtration through a small-pore mesh sieve. In a preferred embodiment, the filtration refers to first performing a primary filtration of the digested tissue fluid through a sterile 200μm large-pore sieve to remove incompletely digested tissue, and then performing a secondary filtration of the tissue lysate using a sterile 40μm small-pore mesh sieve to remove as much incompletely digested tissue residue as possible.

[0059] In step (3), since a rotation speed greater than 2000 rpm will cause cell damage, making it difficult for muscle stem cells to maintain their own proliferative activity, the centrifugation speed in this step of the present invention is 500-2000 rpm, which can be 500-800, 800-1000, 1000-1200, 1200-1500, 1500-1800, or 1800-2000; preferably, it is 500-1800 rpm; more preferably, it is 800-1200 rpm; and even more preferably, it is 1000 rpm.

[0060] In step (3), the centrifugation time can be selected according to actual needs, and can be 1-10 min, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 min; in a more preferred embodiment, the digestion time is 4 min.

[0061] (4) Cell inoculation and culture

[0062] The cell pellet obtained by centrifugation was resuspended in DMEM medium and seeded into culture dishes.

[0063] In step (4), the DMEM culture medium contains growth factors, preferably FGF2. The content of the growth factor can be determined according to actual needs, and can be 1-50 ng / ml, or 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 ng / ml; preferably, the amount of FGF2 in the culture medium is 5-20 ng / ml; more preferably, it is 10 ng / ml.

[0064] In a preferred embodiment, the DMEM medium is a medium containing a high concentration of serum. The amount of serum in the medium is 15-30% (v / v), preferably 20% (v / v).

[0065] In step (4), the culture temperature can be selected according to actual needs, such as 30-40℃, for example 30-32℃, 32-34℃, 34-36℃, 36-38℃, 38-40℃; preferably, it is 32-38℃; in a more preferred embodiment, the culture temperature is 37℃.

[0066] In step (4), the culture time can be selected according to actual needs, such as judging whether cells can be harvested or passaged based on cell growth density. Preferably, the present invention allows cell harvesting when the cell density reaches 90% or higher. Typically, reaching 90% cell density requires 3-5 days of culture. In some preferred embodiments, the culture process also includes a step of changing the culture medium. Since stem cells adhere slowly, cell adhesion takes about 2 days. After adhesion, the cells are washed three times with PBS buffer to remove floating impurities, and then cultured in the aforementioned fresh high-serum culture medium to obtain P0 generation cells. The total culture time for P0 generation cells is more than 3 days.

[0067] (5) Transmission

[0068] P0 generation cells were passaged when the density in the culture dish reached 90%, and cultured in a novel muscle stem cell culture medium.

[0069] In step (5), the P0 generation cells are cells cultured for 3 days when the density in the culture dish reaches 90%.

[0070] (6) Transmission

[0071] P0 generation cells were passaged when the density in the culture dish reached 90%, and cultured using a novel muscle stem cell. The cells adhered and grew for 3 days to obtain the first generation (P1) cells. Then, the second generation (P2), third generation (P3), and fourth generation (P4) cells were cultured in the same way.

[0072] This invention further provides a method for preparing cell-cultured meat, comprising the following steps:

[0073] 1) Bovine muscle stem cells were obtained by culturing according to the above method;

[0074] 2) Harvest the bovine muscle stem cells and culture them under suitable conditions for the formation of muscle fibers and texture to obtain cultured meat.

[0075] The present invention further provides a cultured meat, which is obtained by culturing muscle stem cells obtained by the above-described culture method.

[0076] This invention discloses a non-animal-derived hydrolysate containing suitable amino acids and peptides that can promote the proliferation of muscle stem cells in a basal culture medium containing only low levels of serum, and maintain the stemness of in vitro proliferated muscle stem cells.

[0077] FGF2, a member of the fibroblast growth factor family, is composed of 155 amino acids. It promotes cell mitosis and thus cell proliferation, participating in various biological processes. FGF2 is widely present in bovine nerve tissue, pituitary gland, and adrenal cortex. The bovine FGF2 mentioned and used in this invention is derived from in vitro expression and purification in *E. coli* and exhibits very high biological activity.

[0078] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0079] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0080] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques from molecular biology, biochemistry, cell culture, and related fields. These techniques have been thoroughly described in existing literature.

[0081] In the following examples, unless otherwise specified, the amount of each component in the culture medium refers to the concentration or percentage relative to the muscle stem cell culture medium.

[0082] Example 1: Domestic FGF2 replaces imported FGF2

[0083] The imported FGF2 product information used in this embodiment is FGF2-R&D 2099-FB.

[0084] Due to the long supply cycle and high price of imported FGF2, this invention provides a domestic alternative for FGF2, which is essential in cell culture. The main conclusion is drawn by comparing cell growth under two different FGF2 sources. Specifically, primary cultured muscle stem cells were seeded into 96-well plates in three groups: a control group without FGF2 (Ctrl), an imported FGF2 group (FGF2-R&D), and a domestically produced FGF2 group. Each group had three replicates, with 2000 cells seeded per well. After cell attachment, different culture conditions were applied: the control group received DMEM + 20% FBS, the imported FGF2 group received DMEM + 20% FBS + 10 ng / ml FGF2, and the domestically produced FGF2 group received DMEM + 20% FBS + 10 ng / ml FGF2. Cells were cultured for 5 days, and CCK8 cell proliferation was measured on days 1, 3, and 5. After the measurements, the data were processed and plotted.

[0085] The results are as follows Figure 4 As shown, from Figure 4It can be seen that cells without FGF2 proliferate slowly, with a low OD value. The addition of domestic and imported FGF2 can significantly promote the proliferation of muscle stem cells. Furthermore, it can be seen that the promoting effects of domestic and imported FGF2 on cell proliferation are comparable. Therefore, it can be concluded that domestic FGF2 can replace imported FGF2, which is more expensive and has a longer lead time.

[0086] Example 2: Isolation and Culture of Bovine Muscle Stem Cells

[0087] (1) Organize collection

[0088] Collect fresh beef muscle tissue from markets where the slaughter time is less than 3 hours; use a scalpel to remove 0.5 cm of tissue from the surface of the muscle, and soak 10 g of the remaining muscle tissue in a 50 mL centrifuge tube containing 75% alcohol in an ice bath for 2 minutes for preliminary sterilization; then transfer it to a 50 mL centrifuge tube containing 10% (v / v) penicillin-streptomycin bispecific antibody in an ice bath.

[0089] (2) Tissue fragmentation and digestion

[0090] The obtained bovine muscle tissue was transferred to a sterile room for cell separation. The tissue was removed from PBS buffer and placed in a 10cm culture dish. The tissue was then minced using sterile surgical scissors to a size of 0.5mm. 3 (Be careful of mechanical shearing force during the mincing process to prevent cell damage); transfer it to a 50ml centrifuge tube, add twice the volume of trypsin digestion solution (if there is obvious incomplete digestion during tissue digestion, the digested tissue can be divided into two 50ml centrifuge tubes, and then add one volume of trypsin digestion solution for digestion), and transfer it to a 37℃ constant temperature incubator for digestion. During digestion, shake it every 5 minutes (to ensure thorough digestion), and the digestion time is 1 hour; divide the digested tissue fluid into two portions, add an equal volume of DMEM medium containing 20% ​​(v / v) serum to 30ml, and stop digestion;

[0091] (3) Tissue filtration

[0092] After digestion, the tissue lysate was first filtered using a sterile 200μm large-pore screen to remove incompletely digested tissue. Then, the tissue lysate was filtered a second time using a sterile 40μm small-pore screen (the purpose of which is to remove as much undigested tissue residue as possible). The filtered cell suspension was then centrifuged at 1000 rpm for 4 min.

[0093] (4) Cell inoculation

[0094] The cell pellet obtained by centrifugation was slowly resuspended in DMEM medium (high serum medium) containing 20% ​​(v / v) serum and 10 ng / ml FGF2 growth factor, and then seeded into 10 cm culture dishes and transferred to a 37°C incubator for culture.

[0095] (5) Cell culture expansion

[0096] Stem cells adhere slowly, so cell adhesion takes 48 hours. After adhesion, wash three times with PBS buffer to remove floating impurities, then add the above-mentioned fresh high-serum culture medium for culture to obtain P0 generation cells. The total culture time for P0 generation cells is more than 3 days.

[0097] Table 1

[0098]

[0099] AN / TN (%) refers to Ammonia Nitrogen / Total Nitrogen (%DH), which is the mass ratio of amino nitrogen to total nitrogen.

[0100] kDa refers to the molecular weight of a polypeptide; its corresponding value represents the percentage of total peptides in yeast hydrolysate of polypeptides within that molecular weight range.

[0101] The information for the two hydrolysates is as follows: Angel CM-YE-01 (item number) and Angel CM-YE-03 (item number).

[0102] In Table 1, hydrolysate #1 and hydrolysate #2 are powdered products obtained by using baker's yeast cultured with high-density fermentation control technology as raw material, and by bio-directed degradation and membrane filtration technology, followed by concentration and spray drying.

[0103] The preparation of the culture medium containing non-animal-derived hydrolysate in this embodiment is as follows: First, weigh the appropriate amount of hydrolysate and dissolve it in DMEM medium. Then, add 0.5% (v / v) serum and 10 ng / ml domestic FGF2 to prepare the culture medium containing non-animal-derived hydrolysate. During the passage of the first-generation cells, after cell counting, take 300,000 cells and divide them into three groups of 100,000 cells each. Seed them into three wells of a 6-well plate. The culture medium for this process is DMEM medium containing 20% ​​serum and 10 ng / ml FGF2. After the cells adhere, add the corresponding culture medium to the three wells. The composition of the five groups of culture media is as follows (except for DMEM, all components are described based on DMEM):

[0104] DMEM + 20% FBS + 10 ng / ml FGF2 (control group, Ctrl);

[0105] DMEM + 0.5% FBS + 1 mg / ml hydrolysate #1 + 10 ng / ml FGF2 (domestic) (hydrolysate 1);

[0106] DMEM + 0.5% FBS + 1 mg / ml hydrolysate #2 + 10 ng / ml FGF2 (domestic) (hydrolysate 2).

[0107] (6) Transmission

[0108] P0 generation cells were passaged when the culture dish density reached 90%. After cell counting, 300,000 cells were collected and divided into three groups of 100,000 cells each. These were seeded into three wells of a 6-well plate and cultured in a medium containing non-animal-derived hydrolysate. Cells adhered and grew for 3 days to obtain the first generation (P1) cells. The second (P2), third (P3), and fourth (P4) generation cells were then cultured using the same method. Micrographs of bovine muscle stem cells cultured using different hydrolysates for four generations (P1–P4) are shown below. Figure 4 As shown in the figure, both hydrolysate 1 and hydrolysate 2 can achieve cell proliferation and passage. The culture medium containing hydrolysate 1 can maintain cell proliferation for multiple generations and for a long time, which is on par with the proliferation of control cells. The culture medium containing hydrolysate 2 can basically maintain normal cell proliferation and passage.

[0109] (7) Cell growth process and state detection

[0110] Stem cell characterization: Stem cell characterization of P2 cells was performed using PAX7, a classic protein marker for muscle stem cells. The procedure was as follows: P1 generation cells were passaged when the cell density reached 90%. After cell counting, 20,000 cells were transferred to 24-well plates with slides. Once the cells adhered, protein detection was performed using classic immunofluorescence techniques for antibody incubation and staining. Finally, fluorescence microscopy was used for examination. The results are shown below. Figure 2 As shown, by Figure 2 It can be seen that the proportion of PAX7 positive cells reached more than 90%, indicating that the obtained cells were all muscle stem cells, and the cell isolation and extraction were successful.

[0111] Flow cytometry stemness identification: The stemness of P1 generation cells was identified using the cell surface markers FITC-CD56 and APC-CD29. The procedure was as follows: When the P1 generation cells reached 90% confluence in the culture dish, 200,000 cells were collected and divided into two groups of 100,000 cells each. Cell staining was performed using classic flow cytometry methods. One group was left unstained, while the other group received two different antibodies. After incubation and staining, the cells were analyzed using a flow cytometer. The results are shown below. Figure 3 As shown, by Figure 3It can be seen that the proportion of CD56 and CD29 double-positive cells in the P2 generation reached 85.7%, further indicating that the obtained cells are muscle stem cells.

[0112] In summary, the in vitro culture method, culture medium, and application of muscle stem cells of the present invention effectively overcome the various shortcomings of the prior art and have high industrial application value.

[0113] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. Use of non-animal derived hydrolysate as serum replacement in culturing muscle stem cells; the non-animal derived hydrolysate is yeast hydrolysate; the mass percentage of polypeptides with molecular weight in the range of greater than 2 KDa, 1-2 kDa, 400 Da-1 kDa, 180-400 Da, less than 180 Da in total polypeptides is 0.5-0.8%, 0.8-15%, 15-38%, 40-60%, 10-15% respectively; the mass ratio of amino nitrogen to total nitrogen in the yeast hydrolysate is 25-50%.

2. Use according to claim 1, characterized in that, The yeast hydrolysate is baker's yeast hydrolysate.

3. A muscle stem cell culture medium, characterized by, The muscle stem cell culture medium comprises a basal medium, the non-animal derived hydrolysate as described in the use of any one of claims 1-2 and FGF2; the concentration of the non-animal derived hydrolysate in the muscle stem cell culture medium is 0.1-20 mg / ml based on the dry weight of the non-animal derived hydrolysate; the concentration of the FGF2 in the muscle stem cell culture medium is 1-50 ng / ml; the basal medium is selected from at least one of DMEM, RPMI 1640, MEM, DMEM / F12, F10, CD293, medium 231, medium 106 and a basal medium modified based thereon.

4. The muscle stem cell culture medium of claim 3, wherein, The muscle stem cell culture medium further comprises serum, and the volume percentage of the serum in the muscle stem cell culture medium is 0.5-2%.

5. Use of the muscle stem cell culture medium as described in any one of claims 3-4 in culturing muscle stem cells in vitro.

6. Use according to claim 5, characterized in that, The non-animal derived hydrolysate is used to promote the proliferation of muscle stem cells in vitro and to maintain the stemness of muscle stem cells.

7. A method for culturing muscle stem cells in vitro, characterized by, The muscle stem cells are cultured in vitro using the muscle stem cell culture medium as described in any one of claims 3-4.

8. The culture method according to claim 7, characterized by, The method further comprises a step of isolating muscle stem cells.

9. The culture method as claimed in claim 8, characterized by, The culture method comprises: (1) Tissue collection Muscle tissue is collected and subjected to sterilization treatment; (2) Tissue crushing and digestion The muscle tissue is crushed and then subjected to digestion by adding protease to obtain a digestion solution; (3) Tissue filtration The digestion solution is filtered and centrifuged to obtain a cell precipitate; (4) Cell inoculation and culture The cell precipitate is resuspended using the muscle stem cell culture medium as described in any one of claims 3-4 and inoculated into a culture dish for culture.

10. A method for preparing cell-cultured meat, characterized in that, The method comprises the following steps: 1) Muscle stem cells are obtained by culturing according to the in vitro culture method described in any one of claims 7-9; 2) The muscle stem cells are harvested and cultured under suitable conditions for forming muscle fibers and texture to obtain cell culture meat.

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