A culture medium and its application, and a method for in vitro isolation and culture of yak skeletal muscle satellite cells

Abstract

The present invention discloses a culture medium and its application, as well as a method for in vitro isolation and culture of yak skeletal muscle satellite cells, which relates to the technical field of cell culture. The culture medium is composed of the following raw materials: fetal bovine serum with a mass fraction of 9-13%, penicillin at 95-105 U / mL, streptomycin at 95-105 μg / mL, bamboo leaf flavonoids at 5-12 μmol / L, apigenin at 20-30 μmol / L, and the balance is DMEM. The present invention mainly adds appropriate amounts of bamboo leaf flavonoids and apigenin to the culture medium. The two cooperate synergistically, significantly improving the proliferation ability of bovine skeletal muscle satellite cells, and further applying it to the in vitro isolation and culture of yak skeletal muscle satellite cells, establishing a complete and unique sterile isolation, purification, and culture system for yak skeletal muscle satellite cells, providing methods and approaches for a series of future research carried out through yak skeletal muscle satellite cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and particularly relates to a culture medium and its application, and a method for in vitro isolation and culture of yak skeletal muscle satellite cells. Background Art

[0002] In the technical field of cell culture, as the basic environment for cell growth and proliferation, the culture medium has a crucial impact on the growth state, proliferation ability and functional characteristics of cells. Especially for specific types of cells, such as bovine skeletal muscle satellite cells like yak skeletal muscle satellite cells, the optimization of the culture medium is a key link in the research.

[0003] Yak, as a unique livestock species living in plateau areas, its skeletal muscle satellite cells have unique biological characteristics and potential application values. Yak skeletal muscle satellite cells are good models for exploring the process of yak muscle formation. Establishing an in vitro isolation and culture system for skeletal muscle satellite cells is of guiding significance for exploring the growth and development process of yak muscle. Skeletal muscle satellite cells are usually in a quiescent state. After activation, they enter the cell cycle, fuse with existing myofibers to maintain the stem cell pool, so as to ensure an effective cell turnover. However, when the muscle is damaged, the activity of skeletal muscle satellite cells will be stimulated by the environment and increase, and they will start to divide, proliferate and fuse with each other to form myotubes, and then form new myofibers, participating in the regeneration of muscle tissue, thereby improving the tenderness and taste of meat products of related livestock and poultry. The organizational structure, quantity and size of myofibers formed by the proliferation and differentiation of skeletal muscle cells are also important factors determining the quality of meat of meat-producing animals.

[0004] At present, the research on yak skeletal muscle is mainly limited to the analysis of the organizational structures of different yak skeletal muscles, and the research on the apoptosis process of skeletal muscle cells after yak slaughter, etc. Although these researches provide a certain basis for us to understand the characteristics and functions of yak muscle tissue, there are few reports on exploring the mechanism of yak muscle formation based on skeletal muscle satellite cells. As a key cell type for muscle regeneration and repair, the mechanism of its role in the growth and development process of yak muscle remains to be further explored.

[0005] In the existing cell culture technology, although there are already a variety of culture medium formulations widely used in the culture of different types of cells, when these formulations are applied to yak skeletal muscle satellite cells, there are often problems such as low proliferation efficiency and poor cell state. Therefore, it is particularly important to develop a culture medium optimized for the characteristics of bovine skeletal muscle satellite cells including yak skeletal muscle satellite cells. In addition, obtaining highly differentiated yak skeletal muscle satellite cells quickly, simply and efficiently not only provides experimental materials for studying the regulation mechanism of the growth and development of yak muscle tissue, but also lays a theoretical foundation for further revealing the unique mechanism and potential application value of yak muscle formation. SUMMARY OF THE INVENTION

[0006] To solve the above problems, the present invention provides a culture medium and its application, as well as a method for in vitro isolation and culture of yak skeletal muscle satellite cells.

[0007] In the first aspect, the present invention provides a culture medium, which is composed of the following raw materials:

[0008] Fetal bovine serum with a mass fraction of 9 - 13%, penicillin at 95 - 105 U / mL, streptomycin at 95 - 105 μg / mL, bamboo leaf flavonoids at 5 - 12 μmol / L, apigenin at 20 - 30 μmol / L, and the balance being DMEM.

[0009] Further, the concentration of bamboo leaf flavonoids in the culture medium is 8.5 μmol / L.

[0010] Further, the concentration of apigenin in the culture medium is 25 μmol / L.

[0011] Further, the culture medium is composed of the following raw materials:

[0012] Fetal bovine serum with a mass fraction of 10%, penicillin at 100 U / mL, streptomycin at 100 μg / mL, bamboo leaf flavonoids at 8.5 μmol / L, apigenin at 25 μmol / L, and the balance being DMEM.

[0013] In the second aspect, based on the same inventive concept, the present invention provides an application of the culture medium according to any one of the first aspect in the in vitro isolation and culture of bovine skeletal muscle satellite cells.

[0014] Further, the bovine skeletal muscle satellite cells include yak skeletal muscle satellite cells.

[0015] In the third aspect, based on the same inventive concept, the present invention provides a method for in vitro isolation and culture of yak skeletal muscle satellite cells. As Figure 1 shown, the method for in vitro isolation and culture of yak skeletal muscle satellite cells includes the following steps:

[0016] Select the forelimb skeletal muscle tissue of fetal bovine or yak calves within 2 months of age, and perform separation, sterilization, removal of connective tissue, and mincing to obtain yak skeletal muscle tissue blocks;

[0017] Digest the yak skeletal muscle tissue blocks with pronase, terminate the digestion with a complete culture medium after digestion, and pass through a cell sieve to obtain a filtrate;

[0018] Centrifuge the filtrate and inoculate the obtained resuspended cells into a cell culture dish for adherent culture to obtain purified primary yak skeletal muscle satellite cells;

[0019] The yak skeletal muscle satellite cells are proliferated and cultured using the culture medium described in any item of the first aspect to obtain yak skeletal muscle satellite cells.

[0020] Furthermore, the complete culture medium is composed of the following raw materials:

[0021] Horse serum with a mass fraction of 10%, penicillin at 100 U / mL, streptomycin at 100 μg / mL, amphotericin B at 0.25 μg / mL, and the balance being DMEM.

[0022] The above technical solutions provided in the embodiments of the present invention have at least the following advantages compared with the prior art:

[0023] The embodiments of the present invention provide a culture medium and its application, as well as a method for in vitro isolation and culture of yak skeletal muscle satellite cells. The present invention mainly adds appropriate amounts of bamboo leaf flavonoids and apigenin to the culture medium, and the two cooperate synergistically, significantly improving the proliferation ability of bovine skeletal muscle satellite cells, and further applying it to the in vitro isolation and culture of yak skeletal muscle satellite cells, establishing a complete and unique sterile isolation, purification, and culture system for yak skeletal muscle satellite cells, providing methods and approaches for a series of future research carried out through yak skeletal muscle satellite cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings here are incorporated into the specification and constitute a part of this specification, showing embodiments in line with the present invention, and are used together with the specification to explain the principles of the present invention.

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a schematic flow chart of a method for in vitro isolation and culture of yak skeletal muscle satellite cells provided in the embodiments of the present invention.

[0027] Figure 2 It is the P0 generation of yak skeletal muscle satellite cells obtained in the in vitro isolation and culture of yak skeletal muscle satellite cells in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or prepared by existing methods. For example, high-glucose DMEM medium from Shanghai Sheng'er Biotechnology Co., Ltd. can be directly selected as a commercially available product such as DMEM; at the same time, for the steps and parameters involved, without special limitations or specific descriptions, they can be carried out according to the preparation process steps and parameters disclosed in existing technologies such as CN117821377A - A proliferation medium for maintaining the differentiation potential of bovine skeletal muscle satellite cells and its application, or directly carried out according to the instructions of existing equipment. The present invention document will not elaborate one by one.

[0030] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually determined according to national standards. If there is no corresponding national standard, they are carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0031] Example 1

[0032] This example provides a culture medium, which is composed of the following raw materials:

[0033] Fetal bovine serum with a mass fraction of 10%, penicillin at 100 U / mL, streptomycin at 100 μg / mL, bamboo leaf flavonoids at 8.5 μmol / L, apigenin at 25 μmol / L, and the balance being DMEM.

[0034] Example 2

[0035] This example provides a culture medium, which is composed of the following raw materials:

[0036] Fetal bovine serum with a mass fraction of 9%, penicillin at 95 U / mL, streptomycin at 95 μg / mL, bamboo leaf flavonoids at 5 μmol / L, apigenin at 20 μmol / L, and the balance being DMEM.

[0037] Example 3

[0038] This example provides a culture medium, which is composed of the following raw materials:

[0039] Fetal bovine serum with a mass fraction of 13%, penicillin at 105 U / mL, streptomycin at 105 μg / mL, bamboo leaf flavonoids at 12 μmol / L, apigenin at 30 μmol / L, and the balance being DMEM.

[0040] Comparative Example 1

[0041] This example provides a culture medium, specifically the culture medium provided in Example 1 of the prior art CN117821377A.

[0042] Comparative Example 2

[0043] This example provides a culture medium, which is only different from Example 1 in that: apigenin and bamboo leaf flavonoids are not added (i.e., the addition amounts of apigenin and bamboo leaf flavonoids are both 0 μmol / L); the remaining steps and parameters are the same.

[0044] Comparative Example 3

[0045] This example provides a culture medium, which is only different from Example 1 in that: apigenin is not added (i.e., the addition amount of apigenin is 0 μmol / L); the remaining steps and parameters are the same.

[0046] Comparative Example 4

[0047] This example provides a culture medium, which is only different from Example 1 in that: bamboo leaf flavonoids are not added (i.e., the addition amount of bamboo leaf flavonoids is 0 μmol / L); the remaining steps and parameters are the same.

[0048] Test Example 1

[0049] In this example, referring to the test method in Example 2 of the prior art CN117821377A, the culture media provided in the above Examples 1 to 3 and Comparative Examples 1 to 4 were examined for their respective effects on the in vitro culture and proliferation of bovine skeletal muscle satellite cells.

[0050] Test method: Take bovine skeletal muscle satellite cells at passage P3, inoculate them into a 6-well plate at 1×10 5 / well, add 2 mL of each of the above 7 culture media respectively, and culture for five days, and change the culture medium once on the third day. On the fifth day of culture, observe the cell proliferation situation, digest the cells with 0.25% trypsin at 37°C for 5 min, wash the cells thoroughly three times with PBS buffer, then use a cell counter to count the cells, record the cell number, and use the number of cultured cells obtained with the culture medium provided in Comparative Example 1 as the basic control to calculate the cell amplification multiples of the culture media provided in Examples 1 to 3 and Comparative Examples 2 to 4. The test results are shown in Table 1.

[0051] Table 1

[0052]

[0053] As can be seen from Table 1, under the same culture conditions, the culture media provided in Examples 1 to 3 of the present invention can significantly increase the number of cultured bovine skeletal muscle satellite cells, and the effect is significantly better than that of the culture media provided in Comparative Examples 1 to 4.

[0054] Test Example 2

[0055] In this example, the culture medium provided in Example 1 was further applied to the in vitro isolation and culture of yak skeletal muscle satellite cells, which specifically included the following processes:

[0056] (1) Yak skeletal muscle sampling and treatment: Select the radial carpal extensor muscle of the dorsolateral group from the forelimb skeletal muscle of fetal cattle or yaks within 2 months of age. Immerse the sample in 75% alcohol for 10 - 15 min, wash it 3 times with PBS or normal saline containing antibiotics, put the sample into PBS containing antibiotics, transport it on ice, and quickly transfer it to the cell room.

[0057] (2) Isolation and purification: Sterilely collect the forelimb skeletal muscle tissue of yak calves, place it in 75% ethanol and PBS containing 2% double antibiotics and wash it several times. Remove visible connective tissues such as blood vessels, fat, and fascia in pre-cooled PBS. Add an equal volume of DMEM containing antibiotics to a sterilized small glass bottle, and cut it into tissue blocks of 1 mm 3 in size. Transfer the tissue blocks to a sterile centrifuge tube, centrifuge to remove the supernatant. Add three volumes of 1 mg / mL pronase and digest at 37°C for 1 h, shaking and mixing evenly every 10 min during this period. After digestion, terminate the digestion with an equal volume of complete culture medium (DMEM + 10% horse serum + 100 U / mL penicillin + 100 μg / mL streptomycin + 0.25 μg / mL amphotericin B), and pass through a 40μm cell sieve in sequence. Centrifuge the filtrate at 1000 r / min for 10 min, resuspend the cells and inoculate them into a T25 / T7 cell culture dish. Then place it in an incubator with a volume fraction of 5% CO2 and 37°C for adherent culture for 1 h, and then aspirate the supernatant into a new dish, denoted as P01. After culturing the P01 cells for 18 - 24 h, transfer the supernatant again, denoted as P02. Among them, the P01 and P02 cells are the purified skeletal muscle satellite cells. Change the medium for the first time after 48 h of culture, and then change the complete culture medium every 2 d.

[0058] (3) Subculture and cryopreservation: When the cells in the culture dish grow to 70% - 80% confluence, subculture them at a ratio of 1:2, change the medium every other day, and observe under an inverted fluorescence microscope. For cryopreservation of satellite cells, resuspend the digested cells with cell cryopreservation solution (90% FBS + 10% dimethyl sulfoxide), pipette and mix well, then add them into cryotubes and place them in a programmable freezing container at -80°C overnight. After taking them out, store them in liquid nitrogen for future use. When thawing, take out the cells from liquid nitrogen, immediately put them into a 37°C water bath for rapid thawing, centrifuge to remove the supernatant, and resuspend them with fresh complete medium and transfer them into a new culture dish.

[0059] (4) Use the medium provided in Example 1 to perform proliferation culture on the above yak skeletal muscle satellite cells to obtain yak skeletal muscle satellite cells.

[0060] The yak skeletal muscle satellite cells obtained in this example were observed under an inverted fluorescence microscope. The freshly isolated satellite cells were spherical, with a small volume and strong refraction; and during in vitro culture, the high undifferentiated state of satellite cells was maintained, and their huge differentiation potential was retained, as Figure 2 shown.

[0061] In summary, the embodiments of the present invention provide a medium and its application, as well as a method for in vitro isolation and culture of yak skeletal muscle satellite cells. The present invention mainly adds appropriate amounts of bamboo leaf flavonoids and apigenin to the medium, and the two cooperate synergistically to significantly improve the proliferation ability of bovine skeletal muscle satellite cells, and further apply it to the in vitro isolation and culture of yak skeletal muscle satellite cells, establishing a complete and unique sterile isolation, purification, and culture system for yak skeletal muscle satellite cells, providing methods and approaches for a series of future research carried out through yak skeletal muscle satellite cells.

[0062] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A culture medium, characterized in that, The culture medium is composed of the following raw materials: Fetal bovine serum with a mass fraction of 9 - 13%, penicillin at 95 - 105 U / mL, streptomycin at 95 - 105 μg / mL, bamboo leaf flavonoids at 5 - 12 μmol / L, apigenin at 20 - 30 μmol / L, and the balance being DMEM.

2. The culture medium according to claim 1, wherein, The concentration of the bamboo leaf flavonoids in the culture medium is 8.5 μmol / L.

3. The culture medium according to claim 1, wherein The concentration of the apigenin in the culture medium is 25 μmol / L.

4. The culture medium according to claim 1, characterized in that, The culture medium is composed of the following raw materials: Fetal bovine serum with a mass fraction of 10%, penicillin at 100 U / mL, streptomycin at 100 μg / mL, bamboo leaf flavonoids at 8.5 μmol / L, apigenin at 25 μmol / L, and the balance being DMEM.

5. Use of the culture medium according to any one of claims 1 - 4 in the in vitro isolation and culture of bovine skeletal muscle satellite cells.

6. The application according to claim 5, characterized in that, The bovine skeletal muscle satellite cells include yak skeletal muscle satellite cells.

7. A method for in vitro isolation and culture of yak skeletal muscle satellite cells, characterized in that, The method for the in vitro isolation and culture of the yak skeletal muscle satellite cells comprises the following steps: Select the forelimb skeletal muscle tissue of fetal bovine or yak calves within 2 months of age, isolate, sterilize, remove connective tissue, and cut into pieces to obtain yak skeletal muscle tissue blocks; Digest the yak skeletal muscle tissue blocks with pronase, terminate the digestion with a complete culture medium after digestion, pass through a cell sieve to obtain a filtrate; Centrifuge and separate the filtrate, resuspend the cells and inoculate them into a cell culture dish for adherent culture to obtain purified primary yak skeletal muscle satellite cells; Use the culture medium according to any one of claims 1 - 4 to perform proliferation culture on the yak skeletal muscle satellite cells to obtain yak skeletal muscle satellite cells.

8. The method for in vitro isolation and culture of yak skeletal muscle satellite cells according to claim 7, characterized in that, The complete culture medium is composed of the following raw materials: Horse serum with a mass fraction of 10%, penicillin at 100 U / mL, streptomycin at 100 μg / mL, amphotericin B at 0.25 μg / mL, and the balance being DMEM.

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