A method for extracting salmon muscle satellite cells
By adjusting cell culture medium and optimizing temperature treatment, the problem of difficulty in growing salmon muscle satellite cells is solved, efficient extraction and separation is achieved, providing the basis for cell culture meat, simplifying the process and improving cell viability and growth efficiency.
Patent Information
- Application Number
- CN202310782524.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-29
- Publication Date
- 2025-01-03
- Estimated Expiration
- 2043-06-29
AI Technical Summary
The existing muscle extract and traditional culture medium cannot effectively achieve the growth of salmon muscle satellite cells, limiting the process of their extraction and isolation culture.
By adjusting the cell culture medium and optimizing the proliferation temperature, specific steps include treating salmon muscle tissue in a sterile environment, digesting and resuspension using antibiotics and collagenase-containing medium, digesting and filtration in combination with trypsin, and finally at a specific temperature Cell culture is carried out to ensure cell survival and proliferation.
The efficient extraction and separation of salmon muscle satellite cells was achieved, providing the "seed" cells of cell culture meat, laying the foundation for further cell culture, simplifying the extraction steps and improving cell viability and growth efficiency.
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Figure CN116751742B_ABST
Abstract
Description
Technical field:
[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a method for extracting salmon muscle satellite cells. Background technology:
[0002] With the rapid development of my country's social economy, meat products have been in short supply in recent years. Traditional animal husbandry consumes a lot of resources, causes serious environmental pollution, and also has problems such as animal welfare and animal diseases. The contradictions are becoming increasingly prominent, and there is an urgent need to develop green meat production technology. Cell-cultured meat is a disruptive meat production technology that has emerged in recent years. It aims to use stem cells and tissue engineering to produce agricultural and sideline products without sacrificing animals. To produce cell-cultured meat, you first need to obtain "seed" cells such as muscle satellite cells, adipose stem cells, and mesenchymal stem cells. Among them, the most commonly used are muscle satellite cells, which are the main adult stem cells of muscle.
[0003] Muscle satellite cells, also known as muscle stem cells, are a type of mononuclear stem cells located between the basement membrane and the myocyte membrane of muscle tissue and are generally in a quiescent state. When stimulated or damaged, they will be activated and proliferate and differentiate into myoblasts. One part can fuse with other myoblasts to form new multinuclear myotubes to form muscle fibers; the other part self-renews and replenishes stem cells to maintain their own number stability. Muscle satellite cells can be cultured in vitro after separation and extraction, and proliferate and differentiate to form muscle tissue. In recent years, the extraction and culture of muscle satellite cells of animals such as pigs, cattle, and chickens have been systematically developed. Due to species differences and technical incompatibility, there is no good separation method for the extraction and separation of muscle satellite cells of marine fish, especially salmon.
[0004] Salmon is known as the "Supreme Fish" and "Treasure of the Water". It has delicious meat, good taste, and is rich in nutrients such as Omega-3 unsaturated fatty acids. It is one of the healthiest fish in the world. At present, salmon is in short supply in the market and the price remains at a high level. Salmon muscle satellite cells can be used as "seed" cells for cell-cultured meat, so their extraction and separation methods are also one of the important parts in the development of salmon cell-cultured meat. Therefore, it is of great significance to develop an efficient and stable method for extracting salmon muscle satellite cells. However, the current muscle extract and traditional culture medium cannot achieve the growth of micro cells, which limits the extraction of salmon muscle satellite cells and further separation and culture. Summary of the invention:
[0005] The technical problem to be solved by the present invention is to develop an efficient and stable method for extracting salmon muscle satellite cells, which is of great significance. However, the current muscle extract and traditional culture medium cannot achieve the growth of micro cells, which limits the extraction of salmon muscle satellite cells and further separation and culture.
[0006] To solve the above problems, the present invention achieves the extraction and separation culture of salmon muscle satellite cells by adjusting the cell culture medium and optimizing the proliferation temperature, laying a foundation for further obtaining salmon cell cultured meat.
[0007] To achieve the above object, the present invention is implemented by the following technical scheme: a method for extracting salmon muscle satellite cells, comprising the following steps:
[0008] (1) selecting salmon fry, killing them, dissecting them under a sterile environment, obtaining salmon muscle tissue, and temporarily storing them in L-15 basal medium containing antibiotics;
[0009] (2) Wash the muscle tissue with PBS and cut it into small pieces with scissors;
[0010] (3) Transfer the minced muscle tissue to L-15 basal medium containing antibiotics and collagenase I and digest at room temperature;
[0011] (4) centrifuging the digestion solution;
[0012] (5) Add the pellet after centrifugation into L-15 complete medium, pipette to resuspend, and centrifuge;
[0013] (6) The precipitate was resuspended and inoculated into a 6-well plate and cultured at a temperature range of 15°C to 28°C. Within this temperature range, salmon muscle satellite cells can survive and proliferate, and cells grown at 28°C have better growth.
[0014] Furthermore, in step (3), the amount of collagenase I added is 0.33 mg / mL to 0.5 mg / mL; and the digestion time is 30 min to 45 min. Collagenase hydrolyzes proline in the intercellular matrix, thereby dispersing cells, but too high an enzyme concentration and too long a digestion time will damage cell viability; and a lower concentration of enzyme is difficult to effectively dissociate and release cells. In addition, since the enzyme price is relatively high, appropriately reducing the enzyme concentration and extending the digestion time can also save costs.
[0015] Furthermore, the centrifugation parameters in steps (4) and (5) are 300g for 5 minutes. The centrifugation rate of animal cells is generally 300g, and this centrifugal force can effectively separate cells and impurities. However, too high a speed and too long a centrifugation time will cause cell damage, and a lower speed makes it difficult to separate cells and impurities.
[0016] Furthermore, in step (4), the digestion solution is passed through a 40 μm sieve and then centrifuged. This can be used for cell dispersion and preliminary impurity separation. Removing impurities can achieve efficient and uniform distribution of cells, and dispersing through the sieve can achieve monodisperse characteristics of cells.
[0017] Furthermore, the L-15 complete medium in step (5) contains 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 2ng / mL FGF-2, and 20mM HEPES. Fetal bovine serum is rich in nutrients necessary for cell growth and can promote cell proliferation; penicillin-streptomycin and amphotericin are two antibiotics that can inhibit bacterial and fungal proliferation; FGF-2 acts as a cell growth factor to promote cell proliferation; and HEPES can buffer the pH of the culture medium.
[0018] Furthermore, the L-15 complete medium in step (5) contains 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 10 ng / mL FGF-2, 10 ng / mL EGF, and 20 mM HEPES. EGF can promote cell division and growth, and promote cell absorption of nutrients.
[0019] Furthermore, after centrifugation in step (5), the following step (5-2) is further included: after centrifugation, pancreatic enzyme is added to the precipitate, digested at room temperature, complete medium is added to terminate the digestion, the two digestion solutions are combined, sieved, and centrifuged; after centrifugation, the precipitate is resuspended in L-15 complete medium, centrifuged, and repeated twice. The extracellular matrix is further digested with pancreatic enzyme to release more muscle stem cells.
[0020] Furthermore, the centrifugation parameters in step (5-2) are 300g centrifugation for 5 minutes; the amount of pancreatin added is 0.1% to 0.25%; and the digestion time is 5 minutes to 10 minutes. Further digestion with pancreatin removes the extracellular matrix on the cell surface and obtains more single cells. Since pancreatin has a strong digestion ability and has a greater impact on cell viability, it is necessary to control the amount of enzyme added and the digestion time.
[0021] The beneficial effects of the present invention are: by optimizing the extraction steps and culture medium components, the salmon muscle stem cell extraction steps are simplified, the efficient proliferation of salmon muscle stem cells is achieved, and "seed" stem cells are provided for the subsequent preparation of salmon cell cultured meat. Description of the drawings:
[0022] Figure 1 : Example 1 Cell growth status after 48h of cell culture.
[0023] Figure 2 : Example 2 Cell growth status after 48h of cell culture.
[0024] Figure 3 : Example 3A15℃, B28℃ cell culture for 48h cell growth status.
[0025] Figure 4 : Example 4 Cell growth status after 48h of cell culture.
[0026] Figure 5 : The cells in Example 3 were subcultured (28° C.).
[0027] Figure 6 : Comparative Example 1, cell growth conditions after 48h of cell culture.
[0028] Figure 7 : Comparative Example 2, cell growth conditions after 48h of cell culture. Specific implementation method:
[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] Embodiment 1:
[0031] Select salmon fry of about 10 cm, kill them, dissect them under sterile environment, obtain salmon muscle tissue, and temporarily store them in L-15 basal medium containing antibiotics; wash the muscle tissue with PBS, and cut the muscle tissue into 1 mm pieces with scissors. 3 Transfer the minced muscle tissue to L-15 basal medium containing antibiotics and 0.33 mg / mL collagenase I (5 mL per gram of tissue), and digest at room temperature for 45 minutes; pass the digestion solution through a 40 μm sieve and centrifuge at 300 g for 5 minutes; resuspend the precipitate in L-15 complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 2 ng / mL FGF-2, 20 mM HEPES), centrifuge at 300 g for 5 minutes, and repeat twice; finally, resuspend the precipitate and inoculate it in a 6-well plate, culture at 20°C for 48 hours, and observe whether the cells grow and expand.
[0032] like Figure 1 As shown, after 48 hours of cell plating and culture, salmon muscle stem cells survived and stretched, and the cells took on a spindle shape, consistent with the morphology of muscle cells.
[0033] Embodiment 2:
[0034] Select salmon fry of about 10 cm, kill them, dissect them under sterile environment, obtain salmon muscle tissue, and temporarily store them in L-15 basal medium containing antibiotics; wash the muscle tissue with PBS, and cut the muscle tissue into 1 mm pieces with scissors. 3small pieces; transfer the minced muscle tissue to L-15 basal medium containing antibiotics and 0.33 mg / mL collagenase I (5 mL per gram of tissue), digest at room temperature for 30 minutes; centrifuge the digestion solution at 300g for 5 minutes; discard the supernatant, add L-15 complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5μg / mL amphotericin B, 2ng / mL FGF-2, 20mM HEPES), resuspend by blowing, and centrifuge at 300g for 5 minutes; add 0.25% trypsin (5 mL per gram of tissue), digest at room temperature for 15 minutes, pass the digestion solution through a 40μm sieve, add L-15 complete medium to the filtrate, and centrifuge at 300g for 5 minutes; resuspend the precipitate with L-15 complete medium, centrifuge at 300g for 5 minutes, repeat twice; finally, resuspend the precipitate and inoculate it in a 6-well plate and culture at 20℃ for 48 hours.
[0035] like Figure 2 After 48 hours of cell culture, salmon muscle stem cells survived and stretched, and the cells took on a spindle shape, which was consistent with the morphology of muscle cells. However, the cell digestion time was too long, and there were many impurities and dead cells in the culture plate, and the cell viability was poor.
[0036] Embodiment 3:
[0037] Select salmon fry of about 10 cm, kill them, dissect them under sterile environment, obtain salmon muscle tissue, and temporarily store them in L-15 basal medium containing antibiotics; wash the muscle tissue with PBS, and cut the muscle tissue into 1 mm pieces with scissors. 3 The minced muscle tissue was transferred to L-15 basal medium (5 mL per gram of tissue) containing antibiotics and 0.33 mg / mL collagenase I, and digested at room temperature for 30 min; the digestion solution was centrifuged at 300 g for 5 min, the supernatant was discarded, and L-15 complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 2 ng / mL FGF-2) was added, resuspended by pipetting, and centrifuged at 300 g for 5 min; the supernatant was discarded, and L-15 complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 2 ng / mL FGF-2) was added. The tissue was centrifuged at 300 g for 5 min after being resuspended by pipetting and centrifuged at 300 g for 5 min; 0.25% trypsin (5 mL per gram of tissue) was added and digested at room temperature for 15 min. The digestion solution was passed through a 40 μm sieve, and the filtrate was added to L-15 complete medium and centrifuged at 300 g for 5 min; the precipitate was resuspended in L-15 complete medium and centrifuged at 300 g for 5 min, and the reaction was repeated twice; finally, the precipitate was resuspended and inoculated in a 6-well plate and cultured at 15°C and 28°C for 48 h, respectively.
[0038] like Figure 3As shown in the figure, cells were cultured at 15℃ and 28℃ for 48h, and the cells survived at both temperatures. When cultured at 15℃, due to the lower temperature, some cells shrank into balls, and a few cells slightly stretched, and the cell growth state was not as good as that of 28℃ culture.
[0039] like Figure 5 As shown in the figure, salmon stem cells reached the subculture requirement after two weeks of culture at 28°C, and digestion could achieve two consecutive subcultures. During the culture process, the cell morphology changed from a long spindle to a slender shape.
[0040] Embodiment 4:
[0041] Select salmon fry of about 10 cm, kill them, dissect them under sterile environment, obtain salmon muscle tissue, and temporarily store them in L-15 basal medium containing antibiotics; wash the muscle tissue with PBS, and cut the muscle tissue into 1 mm pieces with scissors. 3 The minced muscle tissue was transferred to L-15 basal medium containing antibiotics and 0.33 mg / mL collagenase I (5 mL per gram of tissue) and digested at room temperature for 30 min; the digestion solution was centrifuged at 300 g for 5 min, the supernatant was discarded, and L-15 complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 10 ng / mL FGF-2, 10 ng / mL EGF), resuspended by blowing, and centrifuged at 300g for 5min; discard the supernatant, add L-15 complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5μg / mL amphotericin B, 10ng / mL FGF-2, 10ng / mL EGF, 20mM HEPES), resuspended by blowing, and centrifuged at 300g for 5min; add 0.25% trypsin (5mL per gram of tissue), digest at room temperature for 15min, pass the digestion solution through a 40μm sieve, add L-15 complete medium to the filtrate, and centrifuge at 300g for 5min; resuspend the precipitate with L-15 complete medium, centrifuge at 300g for 5min, repeat twice; finally, resuspend the precipitate and inoculate it in a 6-well plate, and culture it at 28℃ for 48h.
[0042] like Figure 4 As shown, after 48 hours of cell plating and culture, salmon muscle stem cells survived and stretched, and the cells took on a spindle shape, consistent with the morphology of muscle cells.
[0043] Comparative Example:
[0044] The two main basic culture media reported so far for salmon cell culture are DMEM and MEM.
[0045] Comparative Example 1:
[0046] Select salmon fry of about 10 cm, kill them, dissect them under sterile environment, obtain salmon muscle tissue, and store them in DMEM basal medium containing antibiotics; wash the muscle tissue with PBS, and cut the muscle tissue into 1 mm pieces with scissors. 3 Transfer the minced muscle tissue to DMEM basal medium containing antibiotics and 0.33 mg / mL collagenase I (add 5 mL for each gram of tissue) and digest at room temperature for 45 minutes; pass the digestion solution through a 40 μm sieve and centrifuge at 300 g for 5 minutes; resuspend the precipitate in DMEM complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 2 ng / mL FGF-2, 20 mM HEPES), centrifuge at 300 g for 5 minutes, and repeat twice; finally, resuspend the precipitate and inoculate it in a 6-well plate, culture at 20°C for 48 hours, and observe whether the cells grow and expand.
[0047] like Figure 6 As shown, using DMEM as the basal culture medium for cell culture for 48 hours, it can be seen that salmon muscle stem cells do not survive in this culture medium, indicating that this culture medium is not suitable for the culture of salmon muscle stem cells under the culture conditions described in this article.
[0048] Comparative Example 2:
[0049] Select salmon fry of about 10 cm, kill them, dissect them under sterile environment, obtain salmon muscle tissue, and temporarily store them in MEM basal medium containing antibiotics; wash the muscle tissue with PBS, and cut the muscle tissue into 1 mm pieces with scissors. 3 Transfer the minced muscle tissue to MEM basal medium containing antibiotics and 0.33 mg / mL collagenase I (add 5 mL for each gram of tissue) and digest at room temperature for 45 minutes; pass the digestion solution through a 40 μm sieve and centrifuge at 300 g for 5 minutes; resuspend the precipitate in MEM complete medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 2 ng / mL FGF-2, 20 mM HEPES), centrifuge at 300 g for 5 minutes, and repeat twice; finally, resuspend the precipitate and inoculate it in a 6-well plate, culture at 20°C for 48 hours, and observe whether the cells grow and expand.
[0050] like Figure 7 As shown, using MEM as the basic culture medium for cell culture for 48 hours, it can be seen that salmon muscle stem cells shrank into balls in the culture medium and did not grow, indicating that the culture medium is not suitable for the culture of salmon muscle stem cells under the culture conditions described in this article.
[0051] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.
[0052] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.
Claims
1. A method for extracting salmon muscle satellite cells, characterized in that The following steps are involved: (1) selecting salmon fry, killing them, dissecting them under a sterile environment, obtaining salmon muscle tissue, and temporarily storing them in L-15 basal medium containing antibiotics; (2) Wash the muscle tissue with PBS and cut it into small pieces with scissors; (3) Transfer the minced muscle tissue to L-15 basal medium containing antibiotics and collagenase I and digest at room temperature; (4) centrifuging the digestion solution; (5) Add the pellet after centrifugation into L-15 complete medium, pipette to resuspend, and centrifuge; (6) Resuspend the precipitate and inoculate it into a 6-well plate and culture it at 15°C to 28°C; The L-15 complete culture medium contains 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 10 ng / mL FGF-2, 10 ng / mL EGF, and 20 mM HEPES; or the L-15 complete culture medium contains 10% fetal bovine serum, 1% penicillin-streptomycin, 2.5 μg / mL amphotericin B, 2 ng / mL FGF-2, and 20 mM HEPES.
2. The method according to claim 1, characterized in that: In step (3), the amount of collagenase I added is 0.33 mg / mL to 0.5 mg / mL; the digestion time is 30 min to 45 min.
3. The method according to claim 1, characterized in that: The centrifugation parameters in steps (4) and (5) are 300g for 5 minutes.
4. The method according to claim 1, characterized in that: Step (4) The digestion solution is passed through a 40 μm sieve and then centrifuged.
5. The method according to claim 1, characterized in that: After centrifugation in step (5), the following steps (5-2) are also included: after centrifugation, trypsin is added to the precipitate, digested at room temperature, complete culture medium is added to terminate the digestion, the two digestion solutions are combined, passed through a sieve, and centrifuged; after centrifugation, the precipitate is resuspended with L-15 complete culture medium, centrifuged, and repeated twice.
6. The method according to claim 5, characterized in that: The centrifugation parameters in step (5-2) are 300 g for 5 min; the amount of pancreatin added is 0.1% to 0.25%; and the digestion time is 5 min to 10 min.