Culture medium and application thereof, and in-vitro separation and culture method of yak skeletal muscle satellite cells

Abstract

The invention discloses a culture medium and application thereof, and an in-vitro separation and culture method of yak skeletal muscle satellite cells, and relates to the technical field of cell culture, the culture medium comprises the following raw materials: fetal calf serum with mass fraction of 9-13%, penicillin with mass fraction of 95-105 U / mL, streptomycin with mass fraction of 95-105 [mu] g / mL, bamboo leaf flavone with mass fraction of 5-12 [mu] mol / L, apigenin with mass fraction of 20-30 [mu] mol / L, and the balance of DMEM. A proper amount of bamboo leaf flavonoid and apigenin are added into the culture medium and cooperate with each other, so that the multiplication capacity of the cattle skeletal muscle satellite cells is remarkably improved, and the culture medium is further applied to in-vitro separation and culture of the yak skeletal muscle satellite cells; a complete set of complete and specific sterile separation, purification and culture system for the yak skeletal muscle satellite cells is established, and a method and an approach are provided for a series of researches developed through the yak skeletal muscle satellite cells in the future.

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, as well as 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, 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] Currently, 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 studies 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 action of skeletal muscle satellite cells 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: 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.

[0008] Further, the concentration of bamboo leaf flavonoids in the culture medium is 8.5 μmol / L.

[0009] Further, the concentration of apigenin in the culture medium is 25 μmol / L.

[0010] Further, 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.

[0011] 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.

[0012] Further, the bovine skeletal muscle satellite cells include yak skeletal muscle satellite cells.

[0013] 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: 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; 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; 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; Use the culture medium according to any one of the first aspect to perform proliferation culture on the yak skeletal muscle satellite cells to obtain yak skeletal muscle satellite cells.

[0014] Furthermore, 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.

[0015] The above technical solutions provided by the embodiments of the present invention have at least the following advantages compared with the prior art: 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 studies carried out through yak skeletal muscle satellite cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present invention and used together with the specification to explain the principles of the present invention.

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for 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.

[0018] Figure 1 It is a schematic flow chart of a method for in vitro isolation and culture of yak skeletal muscle satellite cells provided by an embodiment of the present invention.

[0019] Figure 2 It is the P0 generation of yak skeletal muscle satellite cells obtained from the in vitro isolation and culture of yak skeletal muscle satellite cells in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention 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 without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0021] Unless otherwise specified, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase 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. This invention document will not elaborate one by one.

[0022] The following will further elaborate on the present invention 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. For the experimental methods without specific conditions noted in the following embodiments, they 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 the conditions recommended by the manufacturer.

[0023] Example 1 This example provides a culture medium, which 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 is DMEM.

[0024] Example 2 This example provides a culture medium, which is composed of the following raw materials: 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 is DMEM.

[0025] Example 3 This example provides a culture medium, which is composed of the following raw materials: 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 is DMEM.

[0026] Comparative Example 1 This example provides a culture medium, specifically the culture medium provided in Example 1 of the existing technology CN117821377A.

[0027] Comparative Example 2 This example provides a culture medium, which is only different from that in 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.

[0028] Comparative Example 3 This example provides a culture medium, which is only different from that in 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.

[0029] Comparative Example 4 This example provides a culture medium, which is only different from that in 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.

[0030] Test Example 1 In this example, the culture media provided in the above Examples 1 to 3 and Comparative Examples 1 to 4 were examined for their effects on the in vitro culture and proliferation of bovine skeletal muscle satellite cells with reference to the test method in Example 2 of the prior art CN117821377A.

[0031] 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, during which the culture medium is changed 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, and 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.

[0032] Table 1 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.

[0033] Test Example 2 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 process: (1) Yak skeletal muscle sampling and treatment: Select the radial extensor carpi muscle in the dorsolateral group from the 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 physiological saline containing antibiotics, put the sample into PBS containing antibiotics, transport it on ice, and quickly transfer it to the cell room.

[0034] (2) Separation and purification: The skeletal muscle tissue of the forelimb of a yak calf was aseptically collected and successively washed several times in 75% ethanol and PBS containing 2% double antibiotics. Visible blood vessels, fats, fascia and other connective tissues were removed in pre-cooled PBS. Equal volume of DMEM containing antibiotics was added to a sterilized small glass bottle, and the tissue was minced into 1 mm 3 tissue blocks. The tissue blocks were transferred to a sterile centrifuge tube, and the supernatant was removed by centrifugation. Three times the volume of 1 mg / mL pronase was added and digested at 37 °C for 1 h, and the mixture was shaken and mixed every 10 min during this period. After digestion, the digestion was terminated with an equal volume of complete medium (DMEM + 10% horse serum + 100 U / mL penicillin + 100 μg / mL streptomycin + 0.25 μg / mL amphotericin B), and passed through a 40 μm cell sieve in sequence. The filtrate was centrifuged at 1000 r / min for 10 min, the cells were resuspended and inoculated into T25 / T7 cell culture dishes. Then it was placed in an incubator with 5% CO2 by volume and 37 °C for adherent culture for 1 h, and then the supernatant was aspirated into a new dish, denoted as P01. After the P01 cells were cultured for 18 - 24 h, the supernatant was transferred again, denoted as P02. Among them, the P01 and P02 cells are the purified skeletal muscle satellite cells. The medium was changed for the first time after 48 h of culture, and then the complete medium was changed every 2 d.

[0035] (3) Subculture and cryopreservation: When the cells in the culture dish grew to 70% - 80% confluence, subculture was carried out at a ratio of 1:2, and the medium was changed every other day, and observed under an inverted fluorescence microscope. For cryopreservation of satellite cells, the digested cells were resuspended with cell cryopreservation solution (90% FBS + 10% dimethyl sulfoxide), mixed well by pipetting, added to cryopreservation tubes, placed in a programmable cooling box at -80 °C overnight, taken out and stored in liquid nitrogen for later use. When resuscitating, the cells were taken out from liquid nitrogen, immediately put into a 37 °C water bath for rapid thawing, the supernatant was removed by centrifugation, and the cells were resuspended with fresh complete medium and inoculated into a new culture dish.

[0036] (4) The yak skeletal muscle satellite cells were proliferatively cultured using the medium provided in Example 1 to obtain yak skeletal muscle satellite cells.

[0037] The yak skeletal muscle satellite cells obtained in this example were observed under an inverted fluorescence microscope. The freshly isolated satellite cells were spherical, small in volume and had 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.

[0038] In summary, 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 studies carried out through yak skeletal muscle satellite cells.

[0039] 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, and 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 rather will conform to the widest 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: The mass fraction is 9~13% fetal bovine serum, 95~105 U / mL penicillin, 95~105μg / mL streptomycin, 5~12μmol / L bamboo leaf flavonoids, 20~30μmol / L apigenin, and the balance is DMEM.

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

3. The culture medium according to claim 1, characterized in that 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: The mass fraction is 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 8.5 μmol / L bamboo leaf flavonoids, 25 μmol / L apigenin, and the balance is DMEM.

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

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

7. A method for isolating and culturing yak skeletal muscle satellite cells in vitro, characterized in that: The in vitro separation and culture method of yak skeletal muscle satellite cells comprises the following steps: Select forelimb skeletal muscle tissue of fetal bovine or yak calves within 2 months of age, separate, sterilize, remove connective tissue and cut into pieces to obtain yak skeletal muscle tissue blocks; Digesting the yak skeletal muscle tissue block with pronase, terminating the digestion with complete medium after digestion, passing through a cell sieve, and obtaining a filtrate; The filtrate is centrifuged and the obtained resuspended cells are inoculated into a cell culture dish for adherent culture to obtain purified primary yak skeletal muscle satellite cells; The yak skeletal muscle satellite cells are proliferated and cultured using the culture medium described in any one of claims 1 to 4 to obtain yak skeletal muscle satellite cells.

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

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