CN111808801A - Method for extracting and culturing pigeon skeletal muscle satellite cells - Google Patents
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Abstract
The invention relates to the technical field of poultry stem cell culture, in particular to a method for extracting and culturing pigeon skeletal muscle satellite cells. The culture medium comprises chicken serum and a basal culture medium; the basic culture medium is a serum-free high-sugar cell culture medium, contains 3-4 g/L of glucose and 0.3-0.7 mg/L of glutathione, and does not contain ferric nitrate. The culture medium can obviously improve the proliferation efficiency and the cell number of the pigeon skeletal muscle satellite cells, and can effectively ensure the dryness maintenance and the regeneration activity of the pigeon skeletal muscle satellite cells obtained by amplification. The extraction and culture method of pigeon skeletal muscle satellite cells provided by the invention can be used for quickly extracting the pigeon skeletal muscle satellite cells from pigeon embryos and performing stable and efficient amplification in vitro, and the prepared pigeon skeletal muscle satellite cells are uniform and stable in shape and large in cell number, and can be widely used in the fields of artificial meat, cell engineering, pigeon muscle development, molecular biology and the like.
Description
Technical Field
The invention relates to the technical field of poultry stem cell culture, in particular to a culture medium of pigeon skeletal muscle satellite cells and an extraction and culture method thereof.
Background
The skeletal muscle satellite cell is a myoblast with differentiation property between a muscle membrane and a basement membrane, and can be activated to differentiate and proliferate after being stimulated or induced, and further fused to form a multinuclear myotube structure. Since the skeletal muscle satellite cell can be directionally differentiated into myoblasts and is involved in various cell development processes, the skeletal muscle satellite cell is commonly used for in vitro research of muscle development regulation, muscle tissue formation processes, muscle injury, self-renewal repair and the like, and is a model cell with wide application. The skeletal muscle satellite cells of different species of animals can be used as model cells for muscle development, formation, injury repair and the like of different animals. At present, the mature model skeletal muscle satellite cells are mainly derived from mammals such as human, rat, mouse and the like. With the development of scientific breeding, bioengineering, and food industry, the demand for avian animal cells has increased.
The pigeon is taken as a food material for both medicine and food, is popular with wide consumption, and is one of key objects of poultry animal research at present. Extraction and culture of pigeon skeletal muscle cells are the most important of meat cultivation in the new food field. The differentiation and proliferation of pigeon skeletal muscle satellite cells are key steps of myoblast fusion and muscle tissue formation, so that pigeon muscles are cultured in vitro, and the pigeon skeletal muscle satellite cells can be stably and rapidly proliferated in the in vitro culture process, and maintain high regeneration activity and dryness which are factors influencing the quality of the pigeon skeletal muscle satellite cells. Therefore, the development of a high-efficiency and high-quality pigeon skeletal muscle satellite cell separation and culture method is of great significance.
Disclosure of Invention
The invention aims to provide a culture medium for pigeon skeletal muscle satellite cells, and the invention also aims to provide an extraction and culture method for the pigeon skeletal muscle satellite cells.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides a culture medium for pigeon skeletal muscle satellite cells, which comprises chicken serum and a basal culture medium; the basic culture medium is a serum-free high-glucose cell culture medium, and comprises 3-4 g/L of glucose and 0.3-0.7 mg/L of glutathione.
Preferably, the medium of the pigeon skeletal muscle satellite cells does not contain ferric nitrate.
Compared with mammals or other poultry, the pigeon skeletal muscle culture method is less, and the pigeon skeletal muscle satellite cell serving as a pigeon muscle stem cell has no culture method with practical reference significance. Due to different nutrition requirements and proliferation and differentiation regulation and control modes, when the general mammalian or other poultry skeletal muscle satellite cell culture medium is used for pigeon skeletal muscle satellite cell culture, higher proliferation speed and regeneration activity cannot be ensured. On the other hand, when the skeletal muscle satellite cells are rapidly proliferated, it is easy to lose the dryness of a part or a large number of cells, and therefore, it is difficult to achieve a high proliferation rate and a stable dryness at the same time. In the research and development process, the invention unexpectedly discovers that compared with bovine serum commonly used for animal cell culture, the pigeon skeletal muscle satellite cells can ensure higher proliferation rate by adopting chicken serum, under the condition, the sugar content in a basic culture medium is properly increased, and a proper amount of glutathione is added to ensure that the culture medium does not contain ferric nitrate, so that the dryness maintenance and higher regeneration activity can be ensured while the higher proliferation rate is ensured.
Specifically, in the culture medium, the volume percentage content of the chicken serum is preferably 8-12%. More preferably 10%. The invention finds that although the chicken serum can provide rich nutrient substances and growth factors for pigeon skeletal muscle satellite cells, the excessively high addition amount of the chicken serum is not beneficial to the maintenance of the dryness and the regeneration activity of the pigeon skeletal muscle satellite cells.
Preferably, the basal medium comprises the following components: 2.5-3.5 g/L of glucose, 0.4-0.6 mg/L of glutathione and 550-650 mg/L of bacterial peptone. The bacterial peptone with the dosage can be better matched with chicken serum by adding the bacterial peptone with the dosage on the basis of high sugar and glutathione, so that the dryness maintenance and the high regeneration activity are ensured while the high proliferation rate is ensured.
More preferably, the basal medium further comprises inorganic salts, vitamins, amino acids and phenol red.
In a preferred embodiment of the present invention, the basal medium is McCoy's5A medium.
Further, the invention provides an application of the culture medium in pigeon skeletal muscle satellite cell culture.
The invention provides an extraction and culture method of pigeon skeletal muscle satellite cells, which comprises the following steps: and culturing the pigeon skeletal muscle satellite cells by using the medium of the pigeon skeletal muscle satellite cells at the temperature of 39-41 ℃.
In the prior art, most of animal cell culture is carried out at 37 ℃, which is recognized as a proper culture temperature in the field of animal cell culture. However, in the process of research, the invention discovers that the pigeon skeletal muscle satellite cells cultured at a temperature slightly higher than 37 ℃ (39-41 ℃) can not affect the proliferation activity of the pigeon skeletal muscle satellite cells, but are more beneficial to the proliferation of the pigeon skeletal muscle satellite cells, and meanwhile, the loss of dryness or the reduction of regeneration activity can not be caused.
The extraction and culture method also comprises the step of extracting the pigeon skeletal muscle satellite cells, and specifically comprises the following steps: digesting the pigeon skeletal muscle tissue by pancreatin, filtering after digestion, and performing low-speed centrifugal separation to obtain the pigeon skeletal muscle satellite cells.
During the process of pancreatin digestion, the digestion degree of pancreatin is effectively controlled, the activity damage of the satellite cells of the pigeon skeletal muscle due to over digestion is prevented, and the preferred digestion conditions for pigeon skeletal muscle tissues are as follows: adopting 0.25% pancreatin, wherein the addition amount is 2mL/g skeletal muscle tissue, and the digestion condition is that the digestion is carried out for 20-30 min at 37 ℃.
The rotation speed of the low-speed centrifugal separation is lower than 1000 rpm. Preferably, the centrifugation is carried out for 3-5min at 600-800 rpm.
The extraction and culture method of the pigeon skeletal muscle satellite cells specifically comprises the following steps:
(1) taking out pigeon embryo after pigeon egg disinfection, after pigeon embryo disinfection, dissecting pigeon thigh and roughly cutting pigeon skeletal muscle part, tearing out outer fascia, and then tearing skeletal muscle into minced shape;
(2) adding 0.25% pancreatin into minced pigeon skeletal muscle tissues according to the proportion of 2mL/g skeletal muscle tissues, digesting for 20-30 min at 37 ℃, adding a cell culture medium to stop digestion, filtering digested mixed liquor by using a 400-mesh filter screen, centrifuging filtrate for 2-5 min at 600-800 rpm, removing supernatant, and obtaining lower-layer precipitates as target pigeon skeletal muscle satellite cell single cells;
(3) placing the skeletal muscle satellite cells obtained in the step (2) in a culture medium of the skeletal muscle satellite cells, and performing 5% CO treatment at the temperature of 40-41 DEG C2Culturing under the condition.
The invention has the beneficial effects that: the invention provides a pigeon skeletal muscle satellite cell culture medium which is used for in-vitro culture of pigeon skeletal muscle satellite cells, can obviously improve the proliferation efficiency and the cell number of the pigeon skeletal muscle satellite cells, can effectively ensure the dryness maintenance and the regeneration activity of the pigeon skeletal muscle satellite cells obtained by amplification, and obviously improves the quantity and the quality of the pigeon skeletal muscle satellite cells cultured in vitro.
The separation, extraction and culture method of the pigeon skeletal muscle satellite cells provided by the invention can be used for quickly extracting the pigeon skeletal muscle satellite cells from pigeon embryos, reducing the damage to the pigeon skeletal muscle satellite cells and performing stable and efficient amplification by using a culture medium of the pigeon skeletal muscle satellite cells. The pigeon skeletal muscle satellite cells prepared by the method have high purity, uniform and stable morphology, stable dryness maintenance, high regeneration activity and high amplification rate, and can be widely used in the fields of artificial meat, cell engineering, pigeon muscle development, molecular biology and the like.
Drawings
FIG. 1 is a graph showing statistics of cell numbers of pigeon skeletal muscle satellite cells cultured in the culture medium of comparative examples 1 and 2 for 72 hours in Experimental example 1 of the present invention; wherein chicken serum _ DMEM represents the medium of comparative example 2, and fetal bovine serum _ DMEM represents the medium of comparative example 1.
FIG. 2 is a morphological diagram of pigeon skeletal muscle satellite cells after 72h of culture in Experimental example 1 of the present invention using the culture media of comparative examples 1 and 2, wherein (a) and (b) are cell morphologies after 72h of culture at 41 deg.C and 37.5 deg.C using the culture medium of comparative example 2, respectively; (c) and (d) cell morphology after 72h of culture at 41 ℃ and 37.5 ℃ using the culture medium of comparative example 1, respectively.
FIG. 3 is a morphological diagram of pigeon skeletal muscle satellite cells after 72h of culture in Experimental example 1 of the present invention using the culture media of example 1, comparative example 2 and comparative example 3; wherein, (a1) and (a2) are cell forms cultured for 72 hours at 37.5 ℃ by using the culture medium of example 1 respectively; (b1) and (b2) are cell forms obtained by culturing the cells at 41 ℃ for 72h in the medium of example 1; (c1) and (c2) are cell morphologies obtained by culturing the cells at 37.5 ℃ for 72 hours in the culture medium of comparative example 3; (d1) and (d2) are cell morphologies obtained by culturing the cells at 41 ℃ for 72 hours in the culture medium of comparative example 3; (e1) and (e2) are cell morphologies obtained by culturing the cells at 37.5 ℃ for 72 hours in the culture medium of comparative example 2, respectively; (f1) and (f2) are cell morphologies obtained by culturing the cells at 41 ℃ for 72 hours in the medium of comparative example 2, respectively.
FIG. 4 is a graph showing statistics on the number of pigeon skeletal muscle satellite cells cultured for 72 hours in Experimental example 1 using the medium of example 1 and comparative examples 2 and 3, wherein a basal medium-41 ℃ represents the culture at 41 ℃ using the medium of example 1, DMEM-41 ℃ represents the culture at 41 ℃ using the medium of comparative example 2, and M199-41 ℃ represents the culture at 41 ℃ using the medium of comparative example 3.
FIG. 5 is a morphological diagram of pigeon skeletal muscle satellite cells after culturing at 41 ℃ for 72h in Experimental example 1 of the present invention using the cell culture media of example 1 and comparative examples 1, 2 and 3; wherein, (a), (b), (c), (d) are cell morphologies of comparative examples 1, 2, 3 and example 1, respectively.
The scales of the microscope pictures in (a1), (b1), (c1), (d1), (e1) and (f1) of the above fig. 2, 5 and 3 are all 250 μm; the microscope picture scale in (a2), (b2), (c2), (d2), (e2), and (f2) of fig. 3 is 100 μm.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The experimental procedures used in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The chicken sera used in the examples below were purchased from solibao under the cat # S9080; penicillin streptomycin mixed solution (100 Xspecial for cell culture) was purchased from Solebao, cat # P1400.
Example 1
This example provides a medium (500mL) for pigeon skeletal muscle satellite cells, comprising the following components: chicken serum 50mL, McCoy's5A (basal medium) 450 mL.
The formula of the McCoy's5A culture medium is shown in Table 1:
TABLE 1 basal Medium composition
Comparative example 1
This comparative example provides a cell culture medium which differs from the cell culture medium of example 1 only in that the basal medium was changed to DMEM medium and 10% chicken serum was replaced with 10% fetal bovine serum.
Comparative example 2
This comparative example provides a cell culture medium which differs from the cell culture medium of example 1 only in that the basal medium is changed to DMEM medium.
Comparative example 3
This comparative example provides a cell culture medium which differs from the cell culture medium of example 1 only in that the basal medium is changed to M199 medium.
Experimental example 1
The culture medium of the above examples and comparative examples was used for the culture of pigeon skeletal muscle satellite cells, and the effect of each medium and culture conditions on the proliferation, dryness and viability maintenance of pigeon skeletal muscle satellite cells was analyzed. The extraction and culture method of pigeon skeletal muscle satellite cells comprises the following steps:
(1) pigeon egg and pigeon embryo disinfection
Selecting well-developed pigeon eggs without damage to shells of the pigeon eggs and obvious dirt which cannot be removed, lightly wiping the eggs with a 75% alcohol cotton ball for disinfection, lightly knocking an air chamber by medical forceps, lifting the shells to remove white membranes attached to the shells, taking out pigeon embryos by elbow forceps, and soaking the embryos in 75% alcohol for 3 seconds.
(2) Dissecting the pigeon embryo thigh and roughly cutting the pigeon skeletal muscle part
Putting the pigeon embryo rinsed with alcohol into a dish poured with PBS solution in advance, and rinsing twice with the PBS solution; sucking the PBS solution, cutting off the thigh part of the pigeon embryo, tearing off the outer fascia, fixing the muscle by using a pair of forceps, tearing the muscle by using an elbow forceps, and tearing the muscle attached to the skeleton on one thigh of the pigeon embryo into a minced shape.
(3) Digested and dispersed into single cells
Pancreatin which does not pass through the muscle tissue is added into the minced pigeon skeletal muscle tissue, and the addition amount of 0.25 percent of pancreatin is 2mL/g of pigeon skeletal muscle tissue; blowing and beating uniformly, digesting for 30min at 37 ℃, adding cell culture medium with twice volume to stop digestion, filtering the digested mixed solution by using a 400-mesh filter screen, centrifuging the filtrate for 5min at 800rpm, sucking the supernatant, and obtaining the lower-layer sediment which is the target pigeon skeletal muscle satellite cell single cell.
(4) Plates and cultures
Resuspending the cell pellet obtained in step (3) in a cell culture medium, and plating the cell suspension on a plate (10 cm)2Petri dish), each dish of plates 106After adding 5mL of cell culture medium (the cell culture medium of example 1 or comparative example 1, 2 or 3, adding penicillin and streptomycin, the working concentration of penicillin is 100U/mL, the working concentration of streptomycin is 0.1mg/mL), uniformly shaking the cross, and placing the mixture into 5% CO at 37.5 ℃ or 41 DEG C2The cell culture box is used for culturing.
Wherein, the cell culture media of comparative example 1 and comparative example 2 are respectively used for carrying out in vitro culture of the pigeon skeletal muscle satellite cells at the culture temperature of 37.5 ℃ and 41 ℃, the statistical result of the cell number of 72h of culture is shown in figure 1, and the cell morphology is shown in figure 2. The in vitro culture of pigeon skeletal muscle satellite cells was carried out using the culture medium of example 1 and comparative examples 2 and 3 at a culture temperature of 41 ℃ at 37.5 ℃ for 72 hours, and the morphology of the cells after culture is shown in FIG. 3, and the statistics of the cell number after culture for 72 hours are shown in FIG. 4. The morphology of pigeon skeletal muscle satellite cells cultured in the cell culture medium of example 1 and comparative examples 1, 2 and 3 at 41 ℃ for 72h is shown in FIG. 5.
The result shows that the temperature of 41 ℃ is the optimal culture temperature of the pigeon skeletal muscle satellite cells, the number of the cells cultured under the condition of 41 ℃ is obviously higher than other culture temperatures, and the cell morphology is uniform and stable; after culturing for 72h at 41 ℃, the number of cells of the pigeon skeletal muscle satellite cultured by the cell culture medium of the example 1 is increased compared with the number of cells cultured by the cell culture media of the comparative examples 1, 2 and 3, and the pigeon skeletal muscle satellite cells cultured by the cell culture medium of the example 1 have good cell growth state and uniform and stable morphology.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A culture medium for pigeon skeletal muscle satellite cells is characterized by comprising chicken serum and a basal culture medium; the basic culture medium is a serum-free high-sugar cell culture medium; the basic culture medium comprises 3-4 g/L of glucose and 0.3-0.7 mg/L of glutathione.
2. The culture medium of claim 1, wherein the culture medium is free of ferric nitrate.
3. The culture medium according to claim 1 or 2, wherein the volume percentage of the chicken serum in the culture medium is 8-12%.
4. The culture medium according to any one of claims 1 to 3, wherein the basal medium comprises the following components: 2.5-3.5 g/L of glucose, 0.4-0.6 mg/L of glutathione and 550-650 mg/L of bacterial peptone; preferably, the basal medium further comprises inorganic salts, vitamins, amino acids and phenol red.
5. The culture medium according to any one of claims 1 to 4, wherein the basal medium is McCoy's5A medium.
6. Use of the medium according to any one of claims 1 to 5 for the cultivation of pigeon skeletal muscle satellite cells.
7. A method for extracting and culturing pigeon skeletal muscle satellite cells is characterized by comprising the following steps: culturing pigeon skeletal muscle satellite cells at 39-41 ℃ by using the culture medium according to any one of claims 1-5.
8. The method of claim 7, comprising: the pigeon skeletal muscle satellite cells are obtained by digesting pigeon skeletal muscle tissues by pancreatin, filtering and carrying out low-speed centrifugal separation after digestion.
9. The method of claim 8, wherein in the process of pancreatin digestion, 0.25% pancreatin is adopted, the addition amount is 2mL/g skeletal muscle tissue, and the digestion condition is that the digestion is carried out for 20-30 min at 37 ℃.
10. The method according to claim 8 or 9, wherein the rotation speed of the low-speed centrifugation is below 1000 rpm; preferably, the low-speed centrifugal separation is performed at 600-800 rpm for 3-5 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114292809A (en) * | 2021-12-31 | 2022-04-08 | 吉林国健生命工程科学技术有限公司 | Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness |
| CN117487745A (en) * | 2024-01-02 | 2024-02-02 | 中国肉类食品综合研究中心 | Pigeon fat precursor cell separation and in-vitro culture method, culture and application |
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| CN104059876A (en) * | 2014-06-18 | 2014-09-24 | 东北农业大学 | Culture method for improving oxidative metabolic capability of chicken skeletal muscle cells |
| CN106190962A (en) * | 2016-07-27 | 2016-12-07 | 华中农业大学 | Culture medium PM+ of a kind of Animal muscles satellite cell and application thereof |
| CN106497872A (en) * | 2016-12-01 | 2017-03-15 | 北京欣博睿丰医疗科技发展有限公司 | Skeletal muscle stem Cells serum free medium and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114292809A (en) * | 2021-12-31 | 2022-04-08 | 吉林国健生命工程科学技术有限公司 | Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness |
| CN117487745A (en) * | 2024-01-02 | 2024-02-02 | 中国肉类食品综合研究中心 | Pigeon fat precursor cell separation and in-vitro culture method, culture and application |
| CN117487745B (en) * | 2024-01-02 | 2024-03-22 | 中国肉类食品综合研究中心 | Pigeon fat precursor cell separation and in-vitro culture method, culture and application |
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| CN111808801B (en) | 2022-09-30 |
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