Micropterus salmoides skeletal muscle cell line and establishment method thereof

Abstract

The invention relates to a micropterus salmoides skeletal muscle cell line and an establishment method thereof, and belongs to the technical field of aquatic organism cells. At present, research on molecular metabolism pathways of fish for regulating and controlling muscle quality is less due to large individual difference of fish, the method for continuously and efficiently culturing the micropterus salmoides cell line comprises the following steps: treating dorsal muscle tissues of micropterus salmoides, sequentially flushing, digesting, stopping digesting and cleaning, and culturing the micropterus salmoides cell line in a culture medium to obtain the micropterus salmoides cell line. Pasting the tissue block into a T25-coated cell culture bottle for standing, adding a complete culture medium to start primary culture, observing the cell emigration condition at the edge of the tissue block, carrying out subculture by using a pancreatin digestion method containing EDTA when the emigrated cells are fused and spread at the bottom of the bottle, and replacing a subculture medium after the subculture exceeds 15 generations, and cryopreserving the cells which are good and stable in culture state for later use, and resuscitating the cells when the cells are used. According to the method, the cell migration efficiency is greatly improved, and a new path is opened up for continuously culturing the skeletal muscle cell line of the micropterus salmoides.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic biological cells, in particular to a largemouth bass skeletal muscle cell line and an establishment method thereof. Background Art

[0002] In aquaculture, due to the large individual variability among aquatic animals (primarily fish), data obtained from different aquatic animal studies often vary significantly within groups, leading to significant deviations between experimental results and actual conditions. As biologically active in vitro materials, cell lines offer advantages that live fish cannot match: cell culture and maintenance do not require large-scale aquaculture equipment, resulting in low costs, uniform cell size, extremely similar genetic backgrounds, controllable experimental conditions, and high reproducibility. Consequently, the use of fish cell lines in aquaculture research is becoming increasingly widespread, serving as important materials and models for research in fields such as fish nutrition, toxicology, physiology, immunology, and endocrinology. Currently, the cultivation of aquatic animal cells primarily focuses on primary cultures, and the reproducibility of experimental results from primary cells is closely related to the source of the aquatic animal. Therefore, establishing a stable and sustainable cell line is crucial for studying nutritional metabolism, immune regulation, and disease prevention in aquatic animals.

[0003] The largemouth bass (Micropterus salmoides), also known as the California bass, belongs to the order Perciformes, family Heliopsidae, genus Micropterus. It originates from the Mississippi River system in California, USA. Since its introduction to Guangdong Province in 1983, it has rapidly become one of my country's major freshwater carnivorous fishes due to its rapid growth, delicate and delicious meat, rich nutritional profile, strong disease resistance, adaptability to a wide temperature range, and short breeding cycles. In recent years, it has been hailed as the "fifth largest fish." Largemouth bass can be highly productive and efficient in both monoculture and polyculture in most waters of my country, with a market price exceeding 25 yuan per kilogram. Its high economic returns make it a key target for adjusting freshwater aquaculture structures and developing high-quality, high-yield, and efficient fisheries.

[0004] In recent years, there has been an increasing number of studies on the muscle quality of largemouth bass, including Li et al., 2020. Enhanced growth performance, muscle quality and liver health of largemouthbass (Micropterus salmoides) were related to dietary small peptides supplementation. Aquaculture nutrition. 26, 2169-2177. The literature on the effects of adding small peptides to feed on muscle quality; Xie et al., 2021. Effects of compounded animal and vegetable protein feeds on growth performance, muscle quality and nitrogen and phosphorus emissions of Micropterus salmoides; Zhong et al., 2023. Proximate compositions evaluation, histology and transcriptome analysis revealed the effects of formulated diets on muscle quality in Micropterus salmoides. Reproduction and Breeding. 3, 50-58. The literature on the effects of different protein sources on muscle quality; and Song et al., 2024. Effects of Five Lipid Sources on Growth, Hematological Parameters, Immunity and Muscle Quality in Juvenile Largemouth Bass (Micropterus salmoides). Animals. 14,781. Literature has examined the effects of different fat sources on muscle quality. However, existing research primarily focuses on epigenetic data of muscle quality, with relatively little research on the molecular metabolic pathways that regulate muscle quality. Furthermore, due to the large individual variability studied, the data reproducibility is low, making it difficult to truly reflect the accuracy of the data.In view of this, establishing a method for efficiently and sustainably culturing largemouth bass muscle cell lines will help to improve our understanding of the molecular regulatory mechanisms regulating largemouth bass muscle quality and lay the foundation for further promoting the theoretical basis for research on fish muscle quality. Summary of the Invention

[0005] In response to the deficiencies in the above-mentioned prior art, the present invention aims to provide a largemouth bass skeletal muscle cell line and a method for establishing the same. By adding sterile serum of largemouth bass to the complete culture medium and combining it with coating treatment of the culture flask, the efficiency of cell migration can be greatly promoted in the primary start-up culture stage, opening up a new path for the efficient and continuous cultivation of largemouth bass skeletal muscle cell lines, thereby effectively solving the problems existing in the background technology.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention discloses a method for establishing a largemouth bass skeletal muscle cell line, comprising the following steps:

[0008] Step 1: Take the dorsal muscle tissue of largemouth bass and pre-treat it;

[0009] Step 2: Cut the dorsal muscle tissue into tissue blocks, rinse with D-PBS solution containing the three antibodies, and then wash with basal culture medium until there is no impurity. Then, add trypsin containing EDTA for digestion. Then, stop the digestion with complete culture medium, wash, and remove the moisture from the tissue blocks.

[0010] Step 3: Place the dried tissue blocks evenly into the coated T25 cell culture flask and place them upright for 6-12 hours and then inverted for 4-8 hours under constant temperature.

[0011] Step 4: Place the coated T25 cell culture flask upright again and add complete medium to cover the tissue block. Start primary culture at a constant temperature. Change the complete medium regularly and observe the migration of cells at the edge of the tissue block.

[0012] Step 5: When the migrated cells are fused and cover the bottom of the coated T25 cell culture flask, they are subcultured using the EDTA-containing trypsin digestion method. When the subculture exceeds 15 generations, the complete medium is replaced with the subculture medium;

[0013] Step 6: Freeze the cells that are in good and stable culture state for future use. When they are needed, take out the frozen cells and perform cell recovery.

[0014] As a further preferred embodiment of the above scheme: in step 1, the step of pre-treating the back muscle tissue comprises:

[0015] Step 101: Place the separated dorsal muscle tissue in a pre-cooled D-PBS solution containing the triple antibody, clean the dorsal muscle tissue, and remove any residual skin tissue;

[0016] Step 102: Wash the cleaned dorsal muscle tissue three times with pre-cooled D-PBS solution containing the third antibody.

[0017] As a further preferred embodiment of the above scheme: in step 2, the D-PBS solution containing the triple antibody is prepared by adding 1 mL of a mixture of penicillin and streptomycin 100× and 1 mL of amphotericin 100× to every 100 mL of PBS solution.

[0018] Preferably, in step 2, the components of the complete culture medium include:

[0019] 100 mL L15 medium, 18% volume fraction of fetal bovine serum, 2% volume fraction of largemouth bass sterile serum, 100 IU of penicillin, 100 IU of streptomycin, 100 IU of amphotericin, 10 ng / mL FGF and 10 ng / mL IGF, 10 mM HEPES.

[0020] Preferably, in step 2, the step of obtaining sterile serum of largemouth bass comprises:

[0021] Step 201: Using a sterile 1 mL syringe, collect blood from the caudal vertebrae of a largemouth bass on a sterile operating table;

[0022] Step 202: The tail vertebrae blood was allowed to stand overnight, the upper serum was aspirated and centrifuged at 4000 rpm for 10 min, and then sterilized by filtering with a 0.22 μm sterile filter to obtain largemouth bass sterile serum, which was stored at -20°C for later use.

[0023] As a further preferred embodiment of the above scheme: in step 3, the distance between each tissue block is no more than 5 mm, and the constant temperature condition is a constant temperature incubator at 28°C.

[0024] As a further preferred embodiment of the above scheme: in step 5, the step of subculturing by trypsin digestion containing EDTA comprises:

[0025] Step 501: Aspirate the complete culture medium in the coated T25 cell culture flask and heat the trypsin containing EDTA to 28°C;

[0026] Step 502: Add 1 mL of 28°C EDTA-containing trypsin to the coated T25 cell culture flask, gently shake until the trypsin covers the bottom of the flask, then aspirate the trypsin, then add 1 mL of 28°C EDTA-containing trypsin, place in a 28°C constant temperature incubator and let stand for 1-3 minutes, and observe cell contraction under a microscope;

[0027] Step 503: When the cells become round but before they become suspended, immediately aspirate all the trypsin containing EDTA, gently tap the bottom of the coated T25 cell culture flask to remove the cells, then add 6 mL of complete medium (3 mL of new medium + 3 mL of old medium), pipette 3 mL into each of two new T25 cell culture flasks, and mark the passage time and passage number on the new T25 cell culture flasks;

[0028] Step 504: When the cells have been passaged for more than 15 generations, the complete culture medium is replaced with a passage medium.

[0029] As a further preferred embodiment of the above scheme: in step 6, the step of freezing the cells in good and stable culture state for standby use comprises:

[0030] Step 601: Cells are seeded into a T75 culture flask. When the cells reach 95% confluency, they are washed once with a calcium- and magnesium-free PBS solution. 3 mL of 0.25% trypsin is then added to the flask, and the flask is tapped to completely detach the cells.

[0031] Step 602: Add 9 mL of complete culture medium to the T75 culture flask, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant;

[0032] Step 603: Resuspend the cells in 2 mL of cell freezing solution to obtain a cell suspension, divide the cell suspension equally into two cell freezing tubes, place them in a programmed cooling box at -80°C overnight, and then transfer them to liquid nitrogen for long-term storage.

[0033] Preferably, in step 6, the cell recovery operation process is specifically as follows:

[0034] Remove the cell cryovial from liquid nitrogen and place it in a 28°C water bath with shaking for 30 to 60 seconds to make the liquid in the cell cryovial appear in an ice-water compound state. Transfer the cell suspension to a 15 mL centrifuge tube containing 7 mL of complete culture medium. Mix well and centrifuge at 1200 rpm for 5 minutes. Aspirate the supernatant after centrifugation, resuspend the cells in complete culture medium and inoculate them into a T25 cell culture flask. Replace with fresh complete culture medium after 24 hours.

[0035] In a second aspect, the present invention discloses a largemouth bass skeletal muscle cell line, which is obtained by any of the above-mentioned establishment methods.

[0036] Compared with the prior art, the advantages of the technical solution of the present invention are:

[0037] 1. The present invention helps to promote the migration rate of cells in the primary start-up culture stage by adding sterile serum of largemouth bass to the complete culture medium. In the primary start-up culture stage, a tissue block attachment method is adopted, and the culture flask is coated with rat tail collagen to prevent the tissue blocks from falling off, thereby further improving the efficiency of cell proliferation and migration. Experimental results show that the method of the present invention can enable muscle cell migration on the 5th day of primary start-up culture, providing a new idea for the continuous and efficient cultivation of largemouth bass skeletal muscle cell lines.

[0038] 2. The method for establishing a largemouth bass skeletal muscle cell line in a sustained and efficient manner based on the present invention lays the foundation for the study of the molecular regulatory mechanism of largemouth bass muscle quality regulation, and at the same time greatly promotes the theoretical foundation of fish muscle quality research. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0040] Figure 1 This is a diagram of myocyte migration on the fifth day of primary start-up culture of the present invention;

[0041] Figure 2 This is a diagram of myocyte migration on the 15th day of primary start-up culture of the present invention;

[0042] Figure 3 This is a picture of the bottom of the flask when the muscle cells are fully covered on the 28th day of primary start-up culture of the present invention;

[0043] Figure 4 This is a cell diagram of the first generation of cell subculture of the present invention;

[0044] Figure 5 This is a cell diagram of the 50th generation of cell subculture of the present invention;

[0045] Figure 6 This is a single cell chromosome photograph of the present invention;

[0046] Figure 7 This is a photo of the cell chromosome karyotype analysis of the present invention;

[0047] Figure 8 These are ROS fluorescence images of the cells of the present invention before and after infection with Edwardsiella; the left column is the control group (not infected with Edwardsiella); the right column is the experimental group (infected with Edwardsiella). DETAILED DESCRIPTION

[0048] In order to make the purpose, 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 in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0049] Reference Figure 1-8 The present invention provides a method for establishing a largemouth bass skeletal muscle cell line, which is as follows:

[0050] 1. Experimental reagents and materials

[0051] (1) Experimental reagents:

[0052] L15 culture medium was purchased from Gibco; fetal bovine serum was purchased from Biological Industries; penicillin-streptomycin mixture was purchased from Hyclone; amphotericin was purchased from Solebro; rat tail collagen type I was purchased from Solebro; 75% alcohol; 0.25% trypsin was purchased from Hyclone; fibroblast growth factor (FGF) was purchased from Gibco; insulin-like growth factor (IGF) was purchased from Gibco; HEPES solution was purchased from Sigma. All experimental reagents without indicating their sources were purchased through regular commercial channels.

[0053] (2) Experimental materials:

[0054] T25 culture dishes, 6-well cell culture plates, bent forceps, inoculation needles, surgical scissors, scalpels, Pasteur pipettes, and other experimental materials whose sources were not indicated were all purchased through conventional commercial channels; and the bent forceps, inoculation needles, surgical scissors, scalpels, etc. used for dissection needed to be sterilized in a high-pressure sterilizer at 121°C for 30 minutes before use.

[0055] (3) Experimental animals:

[0056] Largemouth bass were obtained from healthy juvenile largemouth bass weighing approximately 10 g from Huzhou City, Zhejiang Province, and were temporarily cultured in an indoor recirculating aquaculture system in the laboratory of the Institute of Immunology and Diseases of Yancheng Institute of Technology. The largemouth bass were cultured for 24 h, and double antibiotics (1000 IU / mL penicillin and 1000 IU / mL streptomycin) were added to the culture water.

[0057] (4) Preparation of solutions and culture medium required for the experiment:

[0058] ① Preparation of D-PBS solution containing the three antibodies: add 1 mL of 100× penicillin-streptomycin mixture and 1 mL of 100× amphotericin to every 100 mL of PBS solution.

[0059] ② Preparation of basal culture medium: Add 1 mL of 100× penicillin-streptomycin mixture, 1 mL of 100× amphotericin, and 1 mL of HEPES solution to every 100 mL of L15 culture medium.

[0060] ③ Preparation of sterile serum of largemouth bass: Take about 10g of healthy largemouth bass juveniles, collect blood from the caudal vertebrae of the largemouth bass with a sterile 1mL syringe on a sterile operating table, let the collected caudal vertebrae blood stand at 4℃ overnight, absorb the upper serum, and then centrifuge it at 4000rpm for 10min, and then filter and sterilize it with a 0.22μm sterile filter to obtain sterile serum of largemouth bass, which is stored at -20℃ for later use.

[0061] ④ Preparation of complete culture medium: Every 100 mL of L15 culture medium contains 18% fetal bovine serum, 2% sterile serum of largemouth bass, 100 IU of penicillin, 100 IU of streptomycin, 100 IU of amphotericin, 10 ng / mL FGF and 10 ng / mL IGF, and 10 mM HEPES. The prepared culture medium is sterilized by filtering with a 0.22 μm filter membrane to prepare a complete culture medium for largemouth bass muscle cells.

[0062] ⑤ Preparation of subculture medium: Every 100 mL of L15 medium contains 10% fetal bovine serum, 100 IU of penicillin, and 100 IU of streptomycin. The prepared medium is sterilized by filtering with a 0.22 μm filter membrane to prepare the subculture medium for largemouth bass muscle cells.

[0063] ⑥ Coating T25 cell culture flask: Pipette rat tail collagen solution into T25 cell culture flask, spread it evenly on the bottom with a cell scraper, wait for 30 minutes, rinse the bottom of the T25 culture flask with PBS solution three times, dry it in a sterile operating table, and cover the flask tightly for later use.

[0064] (5) In vitro application of bacterial infection:

[0065] ① Cell treatment:

[0066] The cells were plated in 6-well plates and infected with E. piscicida at an MOI of 2:1 when the cell confluence was about 80%. The cells were centrifuged at 170 g for 6 min to ensure sufficient contact between bacteria and cells. The control group cells were incubated with an equal amount of L15 complete medium.

[0067] ②Probe loading and detection:

[0068] CM-H2DCFDA was diluted 1:1000 with L15 basal culture medium to a final probe concentration of 5 μM. The original culture medium was aspirated and the diluted CM-H2DCFDA was inoculated into a 6-well cell culture plate at 1.5 mL / well. The cells were incubated at 37°C in the dark for 30 min. After incubation, the cells were washed three times with PBS and 2 mL of L15 complete culture medium was added. The cells were observed and photographed under a fluorescence microscope.

[0069] 2. Culture of Largemouth Bass Skeletal Muscle Cell Line

[0070] (1) Isolation of dorsal muscle tissue of largemouth bass skeletal muscle:

[0071] Healthy largemouth bass juveniles weighing approximately 10 g were selected, the surface of the fish body was wiped with 75% alcohol, and the fish body was soaked in 75% alcohol for 10 seconds. The largemouth bass was then moved into a sterile clean bench, the skin of the fish was scraped open with sterile elbow tweezers, and the dorsal muscle of the largemouth bass was removed with surgical scissors and a scalpel. The separated dorsal muscle tissue was placed in a pre-cooled D-PBS solution containing triple antibodies, the dorsal muscle tissue block was cleaned, the residual skin tissue in the dorsal muscle tissue was removed, and then the dorsal muscle tissue was washed three times with a pre-cooled D-PBS solution containing triple antibodies.

[0072] (2) Digestion and inoculation of dorsal muscle tissue:

[0073] Cut the dorsal muscle tissue of largemouth bass into 1mm 3 The tissue blocks were rinsed twice with D-PBS solution containing triple antibodies (i.e., penicillin, streptomycin, and amphotericin), then placed in a 15 mL centrifuge tube, digested with 0.25% EDTA-containing trypsin at room temperature for 5 min, and complete culture medium was added to the centrifuge tube to terminate the digestion, followed by washing twice with complete culture medium; the liquid-free tissue blocks were then evenly attached to the coated T25 cell culture flask using an inoculation needle, with the spacing between each tissue block not exceeding 5 mm. The coated T25 cell culture flask was placed in a constant temperature incubator at 28°C for 6 h, and then the coated T25 cell culture flask was inverted for 6 h.

[0074] (3) Primary start-up culture:

[0075] After re-positioning the coated T25 cell culture flask upright, slowly add 3-5 mL of complete culture medium to cover the tissue block. Place the flask in a 28°C constant temperature incubator to start primary culture. Replace the complete culture medium every two days. Observe the edge of the tissue block every day to check for cell migration at the edge of the tissue block. Be as gentle as possible during the operation to prevent the tissue block from falling. Take photos and record regularly.

[0076] (4) Subculture:

[0077] When the migrated cells are fused and cover the bottom of the coated T25 cell culture flask, they are subcultured using the 0.25% trypsin digestion method containing EDTA. The specific process of the trypsin digestion method is as follows:

[0078] ① Before digestion, heat the trypsin containing EDTA to 28°C, use a Pasteur pipette to aspirate the complete medium in the coated T25 cell culture flask, and temporarily store the aspirated complete medium aside;

[0079] ② Add 1 mL of 28°C 0.25% trypsin containing EDTA to the coated T25 cell culture flask, shake gently to cover the bottom of the flask, then aspirate the trypsin, add 1 mL of 28°C 0.25% trypsin containing EDTA, and place in a 28°C constant temperature incubator for 1-3 minutes. Observe the cell shrinkage under a microscope.

[0080] ③ When the cells become round but not yet suspended, immediately aspirate all the trypsin, tap the bottom of the coated T25 cell culture flask to remove the cells, then add 6 mL of complete medium (3 mL of new complete medium + 3 mL of the aspirated complete medium), shake gently to mix, and pipette 3 mL into two new T25 cell culture flasks respectively. Mark the passage time and passage number on the new T25 cell culture flasks;

[0081] ④ When the cells have been passaged for more than 15 generations, the complete culture medium was replaced with passage medium.

[0082] (5) Cell freezing and recovery:

[0083] Cell freezing: After the cell culture is in good and stable condition, a large number of cells are frozen for use in subsequent experiments. Specifically, the cells are inoculated into T75 culture flasks. When the cells grow to a confluence of 95%, they are washed once with a PBS solution without calcium and magnesium. Then 3 mL of 0.25% trypsin is added to the T75 culture flask, and the flask is tapped to completely remove the cells. Then 9 mL of complete culture medium is added to the flask, centrifuged at 1200 rpm for 5 minutes, the supernatant is discarded, and the cells are resuspended in 2 mL of cell freezing solution (90% fetal bovine serum, 10% dimethyl sulfoxide) to obtain a cell suspension. The cell suspension is placed in two cell freezing tubes, with 1 mL of cell suspension in each tube, placed in a program cooling box at -80°C overnight, and then transferred to liquid nitrogen for long-term storage.

[0084] Cell recovery: Remove the cell cryovial from liquid nitrogen and place it in a preheated water bath at 28°C for 30-60 seconds with shaking. Transfer the cell suspension to a 15-mL centrifuge tube containing 7 mL of complete medium. Mix thoroughly and centrifuge at 1200 rpm for 5 minutes. Aspirate the supernatant, resuspend the cells in complete medium, and inoculate them into a T25 cell culture flask. Replace the complete medium after 24 hours.

[0085] (6) Experimental results:

[0086] like Figure 1 As shown, when the tissue block adhered to the wall for 5 days, a small number of cells began to migrate out from the edge of the tissue block;

[0087] like Figure 2 As shown, when the tissue block began primary culture for 15 days, the light transmittance around the tissue block increased and the number of cells migrating out increased significantly;

[0088] like Figure 3 As shown, when the primary culture reaches the 28th day, the primary muscle cells have spread over the bottom of the bottle;

[0089] like Figure 4 As shown, the cells of the first passage were slightly disordered and distributed in multiple layers.

[0090] like Figure 5 As shown, the cells were passaged to the 50th generation and cultured in the passage medium. Currently, the cells have been passaged for more than 50 generations, and the muscle cells of each generation have been frozen in liquid nitrogen for subsequent experiments.

[0091] Figure 6 The chromosomes of a single cell are found to be 64 by karyotype analysis (e.g. Figure 7 ), which is inconsistent with the largemouth bass's chromosome number of 48, meaning that largemouth bass muscle cells can proliferate indefinitely.

[0092] like Figure 8 As shown, in order to verify the damage of mitochondria in cells, we used CM-H2DCFDA fluorescent probe to detect the changes in ROS levels in cells 0, 3, 6, 9 and 12 hours after E. piscicida intracellular infection. The results showed that the intracellular ROS content of largemouth bass muscle cells increased with the duration of infection after bacterial infection (refer to Figure 1 In addition, we compared the control group with the infection group and found that the ROS content in the infection group was significantly increased compared with the control group (refer to Figure 8 ).

[0093] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for establishing a largemouth bass skeletal muscle cell line, characterized in that: The following steps are involved: Step 1: Take the dorsal muscle tissue of largemouth bass and pre-treat it; Step 2: Cut the dorsal muscle tissue into tissue blocks, rinse with D-PBS solution containing the three antibodies, and then wash with basal culture medium until there is no impurity. Then, add trypsin containing EDTA for digestion. Then, stop the digestion with complete culture medium, wash, and remove the moisture from the tissue blocks. Step 3: Place the dried tissue blocks evenly into the coated T25 cell culture flask and place them upright for 6-12 hours and then inverted for 4-8 hours at a constant temperature. Step 4: Place the coated T25 cell culture flask upright again and add complete medium to cover the tissue block. Start primary culture at a constant temperature. Change the complete medium regularly and observe the migration of cells at the edge of the tissue block. Step 5: When the migrated cells are fused and cover the bottom of the coated T25 cell culture flask, they are subcultured using the EDTA-containing trypsin digestion method. When the subculture exceeds 15 generations, the complete medium is replaced with the subculture medium; Step 6: Freeze the cells that are in good and stable culture state for future use. When they are needed, take out the frozen cells and perform cell recovery.

2. The method for establishing a largemouth bass skeletal muscle cell line according to claim 1, characterized in that: In step 1, the steps of pre-treating the back muscle tissue include: Step 101: Place the separated dorsal muscle tissue in a pre-cooled D-PBS solution containing the triple antibody, clean the dorsal muscle tissue, and remove any residual skin tissue; Step 102: Wash the cleaned dorsal muscle tissue three times with pre-cooled D-PBS solution containing the third antibody.

3. The method for establishing a largemouth bass skeletal muscle cell line according to claim 2, characterized in that: In step 101 and step 2, the D-PBS solution containing the tertiary antibody is prepared by adding 1 mL of a mixture of penicillin and streptomycin 100× and 1 mL of amphotericin 100× to every 100 mL of PBS solution.

4. The method for establishing a largemouth bass skeletal muscle cell line according to claim 1, characterized in that: In step 2, the complete medium contains: 100 mL L15 medium, 18% volume fraction of fetal bovine serum, 2% volume fraction of largemouth bass sterile serum, 100 IU of penicillin, 100 IU of streptomycin, 100 IU of amphotericin, 10 ng / mL FGF and 10 ng / mL IGF, 10 mM HEPES.

5. The method for establishing a largemouth bass skeletal muscle cell line according to claim 4, characterized in that: In step 2, the steps of obtaining sterile serum of largemouth bass include: Step 201: Using a sterile 1 mL syringe, collect blood from the caudal vertebrae of a largemouth bass on a sterile operating table; Step 202: The tail vertebrae blood was allowed to stand overnight, the upper serum was aspirated and centrifuged at 4000 rpm for 10 min, and then sterilized by filtering with a 0.22 μm sterile filter to obtain largemouth bass sterile serum, which was stored at -20°C for later use.

6. The method for establishing a largemouth bass skeletal muscle cell line according to claim 1, characterized in that: In step 3, the distance between each tissue block is no more than 5 mm, and the constant temperature condition is a constant temperature incubator at 28°C.

7. The method for establishing a largemouth bass skeletal muscle cell line according to claim 1, characterized in that: In step 5, the step of subculturing using the trypsin digestion method containing EDTA includes: Step 501: Aspirate the complete culture medium in the coated T25 cell culture flask and heat the trypsin containing EDTA to 28°C; Step 502: Add 1 mL of 28°C EDTA-containing trypsin to the coated T25 cell culture flask, gently shake until the trypsin covers the bottom of the flask, then aspirate the trypsin, then add 1 mL of 28°C EDTA-containing trypsin, place in a 28°C constant temperature incubator and let stand for 1-3 minutes, and observe cell contraction under a microscope; Step 503: When the cells become round but before they become suspended, immediately aspirate all the trypsin containing EDTA, gently tap the bottom of the coated T25 cell culture flask to remove the cells, then add 6 mL of complete culture medium, pipette 3 mL into each of two new T25 cell culture flasks, and mark the passage time and passage number on the new T25 cell culture flasks. Step 504: When the cells have been passaged for more than 15 generations, the complete culture medium is replaced with a passage medium.

8. The method for establishing a largemouth bass skeletal muscle cell line according to claim 1, characterized in that: In step 6, the step of freezing the cells in good and stable culture state for future use includes: Step 601: Cells are seeded into a T75 culture flask. When the cells reach 95% confluency, they are washed once with a calcium- and magnesium-free PBS solution. 3 mL of 0.25% trypsin is then added to the flask, and the flask is tapped to completely detach the cells. Step 602: Add 9 mL of complete culture medium to the T75 culture flask, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant; Step 603: Resuspend the cells in 2 mL of cell freezing solution to obtain a cell suspension, divide the cell suspension equally into two cell freezing tubes, place them in a programmed cooling box at -80°C overnight, and then transfer them to liquid nitrogen for long-term storage.

9. The method for establishing a largemouth bass skeletal muscle cell line according to claim 8, characterized in that: In step 6, the cell recovery process is as follows: Remove the cell cryovial from liquid nitrogen and place it in a 28°C water bath with shaking for 30 to 60 seconds to make the liquid in the cell cryovial appear in an ice-water compound state. Transfer the cell suspension to a 15 mL centrifuge tube containing 7 mL of complete culture medium. Mix well and centrifuge at 1200 rpm for 5 minutes. Aspirate the supernatant after centrifugation, resuspend the cells in complete culture medium and inoculate them into a T25 cell culture flask. Replace with fresh complete culture medium after 24 hours.

10. A largemouth bass skeletal muscle cell line, characterized in that The cell line is obtained by the establishment method according to any one of claims 1 to 9.

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