Establishment method and application of micropterus salmoides muscle cell line
By using M199 medium-based culture medium and appropriate component configuration, the problem of the lack of largemouth bass muscle cell line establishment was solved, achieving stable and low-cost cell culture and passage, which is suitable for research on largemouth bass muscle cells.
Patent Information
- Application Number
- CN202511340040.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-18
- Publication Date
- 2025-12-05
AI Technical Summary
Currently, a stable muscle cell line for largemouth bass has not been established, making continuous passage and good growth impossible, which limits in vitro research on largemouth bass.
Using M199 medium as a base, a culture medium supplemented with sodium pyruvate, hydroxyethylpiperazine ethanesulfonic acid, β-mercaptoethanol, penicillin, streptomycin, and fetal bovine serum, combined with an appropriate pH value, was used for primary and subcultured largemouth bass muscle cells, and a cryopreservation method was provided.
A stable largemouth bass muscle cell line was established, which can be continuously passaged, has good cell morphology, fast division rate, high adhesion rate, reduces culture cost, is simple and easy to operate, and has good cell viability.
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Figure CN121065083A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of cell culture, in particular to a method for establishing a muscle cell line of largemouth bass and application. BACKGROUND
[0002] Largemouth bass (Micropterus salmoides), also known as California bass, is a carnivorous fish of the Percichthys genus in Perciformes. Largemouth bass is originally from freshwater rivers and lakes in North America, and was introduced to China in the 1970s. Due to its firm texture, delicious taste, and high nutritional value, it has become one of the important economic fish species in China. At present, the annual output of largemouth bass in China is about 800,000 tons, and the growth rate is far ahead of the world. The main production areas are in Guangdong, Zhejiang, Jiangsu and other coastal areas.
[0003] Fish muscle plays an important role in storing glycogen and regulating fat metabolism. Fish muscle, as the main edible part, provides high-quality protein and unsaturated fatty acids. Fish muscle cell lines are the main cell research system in aquatic animal research. Through the study of fish muscle, the influence of feed nutritional components on fish growth and physiological and biochemical effects can be accurately understood, and feed additives can be quickly screened to achieve the research purpose of verifying the precise nutrition of fish in vitro. Therefore, establishing a stable muscle cell line is of great significance for the study of aquatic animal nutrition metabolism, immune regulation and practical application.
[0004] At present, myocardial fibroblast cell lines, rostral cell lines, spinal cord cell lines and brain cell lines have been established in largemouth bass, but largemouth bass muscle cell lines have not been established, and it is impossible to obtain largemouth bass muscle cells that can be continuously passaged and maintain good growth state. Therefore, it is necessary to establish a standardized culture method for largemouth bass muscle cell lines to provide reliable research materials for in vitro research on precise nutrition of largemouth bass.
[0005] Based on this, the present application is proposed. SUMMARY
[0006] The purpose of the present application is to provide a method for establishing a muscle cell line of largemouth bass and application, to solve the problem of lack of construction method of largemouth bass muscle cell line in the prior art.
[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:
[0008] The present application provides a culture solution, which is based on M199 medium and further comprises the following components at the following concentrations:
[0009] 0.5-1.5 mM sodium pyruvate, 90-110 μg / mL hydroxyethylpiperazine ethanesulfonic acid, 90-110 μmol / mL β-mercaptoethanol, 90-110 IU / mL penicillin, 90-110 μg / mL streptomycin, 15-20 vt% fetal bovine serum; the pH value of the culture solution is 7.2-7.4.
[0010] The application provides application of the culture solution in primary and / or subculture of a muscle cell line of Micropterus salmoides.
[0011] The application provides a method for establishing a muscle cell line of Micropterus salmoides, comprising the following steps:
[0012] (1) obtaining muscle of Micropterus salmoides: washing and disinfecting the muscle of Micropterus salmoides to obtain sterile muscle of Micropterus salmoides;
[0013] (2) isolating and culturing primary cells: cutting the sterile muscle of Micropterus salmoides to obtain muscle fragments, mixing the muscle fragments with a culture solution, and culturing at 26-30 °C, replacing the culture solution every 2-3 days to obtain adherent primary cells;
[0014] (3) subculture: when the adherent rate of the adherent primary cells is greater than 80%, digesting the adherent primary cells with a digestion solution to obtain discrete primary cells, mixing the discrete primary cells with a culture solution for subculture to obtain a muscle cell line of Micropterus salmoides;
[0015] The culture solution in steps (2) and (3) is the culture solution.
[0016] Preferably, the washing method in step (1) is soaking the muscle of Micropterus salmoides in PBS buffer containing double antibiotics for 3-5 times, each time for 15-30 s;
[0017] The concentration of the double antibiotics in the PBS buffer is 0-1 vt%;
[0018] The double antibiotics are penicillin and streptomycin;
[0019] The concentration of penicillin in the double antibiotics is 9000-11000 IU / mL, and the concentration of streptomycin is 9000-11000 μg / mL;
[0020] The disinfection method is soaking in 70-80% alcohol for 15-30 s.
[0021] Preferably, the volume of the muscle fragments in step (2) is 0.5-1.5 mm 3The temperature of the culture in step (2) is 26-30 DEG C; the digestive solution in step (3) is a trypsin solution; the concentration of the trypsin solution is 0.2-0.3 wt%.
[0022] The application provides the large-mouth bass muscle cell line obtained by the establishing method.
[0023] The application provides the application of the large-mouth bass muscle cell line in the research of large-mouth bass nutritional metabolism and immune regulation.
[0024] The application provides a freezing method of a large-mouth bass muscle cell line, which comprises the following steps:
[0025] (1) mixing culture solution with freezing culture medium to obtain mixed culture medium;
[0026] (2) mixing the large-mouth bass muscle cell line with the mixed culture medium, freezing at 3-5 DEG C for 25-35 min, then freezing at -18 to -22 DEG C for 1-3 h, and finally freezing at -75 to -85 DEG C overnight to obtain frozen large-mouth bass muscle cell line, which is stored in liquid nitrogen;
[0027] The culture solution in step (1) is the culture solution;
[0028] The large-mouth bass muscle cell line in step (2) is the large-mouth bass muscle cell line.
[0029] Preferably, the volume ratio of the culture solution to the freezing culture medium in step (1) is 1-2:1-2.
[0030] Preferably, the freezing culture medium is composed of M199 culture medium, fetal bovine serum and dimethyl sulfoxide;
[0031] The volume ratio of the M199 culture medium, the fetal bovine serum and the dimethyl sulfoxide is 2-4:1:1.
[0032] The application has the following technical effects and advantages:
[0033] The culture solution used in the application is a M199 culture medium based culture medium, to which sodium pyruvate, hydroxyethylpiperazine ethanesulfonic acid, beta-mercaptoethanol, penicillin, streptomycin and fetal bovine serum are added, and the concentrations of the components are reasonably configured, so that the culture solution provides optimal conditions for the culture of largemouth bass muscle cells and reduces the culture cost. Meanwhile, the culture solution provides a method for establishing a largemouth bass muscle cell line, which is simple and easy to operate, has strong repeatability, and the constructed largemouth bass muscle cell line has good stability, can be continuously passaged and can be ultra-low temperature frozen. The main cell morphology is irregular polygon, fibrocyte-like, the cell division speed is fast, the passage time is short, and the adhesion rate is high. The primary cell culture of largemouth bass muscle in the method takes a short time, and a large number of cells with good activity are obtained, which can be passaged in 8 days. BRIEF DESCRIPTION OF DRAWINGS
[0034] Figure 1 Growth curve of largemouth bass muscle primary cells in different base culture media;
[0035] Figure 2 Growth curve of largemouth bass muscle primary cells in different fetal bovine serum concentrations culture solution;
[0036] Figure 3 Isolated primary cells;
[0037] Figure 4 Adherent primary cells;
[0038] Figure 5 Primary cells with an adhesion rate of 100%;
[0039] Figure 6 Largemouth bass muscle cell line of 15 generations;
[0040] Figure 7 Largemouth bass muscle cell line of 30 generations;
[0041] Figure 8 Largemouth bass muscle cell line of 45 generations;
[0042] Figure 9 Largemouth bass muscle cell line of 60 generations;
[0043] Figure 10 Gel electrophoresis diagram of the PCR amplification fragment of the largemouth bass muscle cell line and the PCR amplification fragment of the largemouth bass muscle tissue. DETAILED DESCRIPTION
[0044] The application provides a culture solution, which is a M199 culture medium based culture medium and further comprises the following components at the following concentrations:
[0045] 0.5-1.5 mM sodium pyruvate, 90-110 μg / mL hydroxyethylpiperazine ethanesulfonic acid, 90-110 μmol / mL β-mercaptoethanol, 90-110 IU / mL penicillin, 90-110 μg / mL streptomycin, 15-20 vt% fetal bovine serum; the pH value of the culture solution is 7.2-7.4;
[0046] The concentration of the sodium pyruvate is preferably 1 mM; the concentration of the hydroxyethylpiperazine ethanesulfonic acid is preferably 100 μg / mL; the concentration of the β-mercaptoethanol is preferably 100 μmol / mL; the concentration of the penicillin is preferably 100 IU / mL; the concentration of the streptomycin is preferably 100 μg / mL; the concentration of the fetal bovine serum is preferably 20 vt%; and the pH value is preferably 7.3.
[0047] The application provides application of the culture solution in primary and / or subculture of a large-mouth bass muscle cell line.
[0048] The application provides a method for establishing a large-mouth bass muscle cell line, comprising the following steps:
[0049] (1) obtaining large-mouth bass muscle: washing and disinfecting the large-mouth bass muscle to obtain sterile large-mouth bass muscle;
[0050] (2) isolating and culturing primary cells: cutting the sterile large-mouth bass muscle to obtain muscle fragments, mixing the muscle fragments with a culture solution, and culturing at 26-30℃, wherein the culture solution is replaced every 2-3 days to obtain adherent primary cells;
[0051] The culturing temperature is preferably 28℃; and the time for replacing the culture solution is preferably every 2 days.
[0052] (3) subculture: when the adherent primary cells have an adherent rate of greater than 80%, the adherent primary cells are digested by a digestion solution to obtain discrete primary cells, the discrete primary cells are mixed with a culture solution for subculture to obtain a large-mouth bass muscle cell line;
[0053] The subculture ratio is 1:1.
[0054] The culture solution in steps (2) and (3) is the culture solution.
[0055] In the application, the washing method in step (1) is soaking the large-mouth bass muscle in a PBS buffer containing double antibodies for 3-5 times, and each time for 15-30 s.
[0056] The concentration of the double antibodies in the PBS buffer is 0-1 vt%.
[0057] The double antibiotic is penicillin and streptomycin;
[0058] The concentration of penicillin in the double antibiotic is 9000-11000 IU / mL, preferably 10000 IU / mL, and the concentration of streptomycin is 9000-11000 μg / mL, preferably 10000 μg / mL;
[0059] The method of cleaning preferably comprises sequentially immersing the muscle of large-mouth bass in PBS buffer solutions with double antibiotic concentrations of 1vt%, 0.5vt%, 0.25vt% and 0vt%, and each immersion lasts for 25s;
[0060] The method of disinfection comprises immersing in 70-80% alcohol for 15-30s;
[0061] The concentration of alcohol is preferably 75%, and the immersion time is 25s.
[0062] In the present application, the volume of the muscle pieces in step (2) is 0.5-1.5mm 3 , preferably 1mm 3 ; the temperature of the culture in step (2) is 26-30℃, preferably 28℃; the digestive solution in step (2) and step (3) is a trypsin solution; the concentration of the trypsin solution is 0.2-0.3wt%, preferably 0.25wt%.
[0063] The present application provides a large-mouth bass muscle cell line obtained by the establishment method.
[0064] The present application provides an application of the large-mouth bass muscle cell line in the research of large-mouth bass nutritional metabolism and immune regulation.
[0065] The present application provides a freezing method of a large-mouth bass muscle cell line, comprising the following steps:
[0066] (1) mixing the culture solution with a freezing culture medium to obtain a mixed culture medium;
[0067] (2) mixing the large-mouth bass muscle cell line with the mixed culture medium, first freezing at 3-5℃ for 25-35min, then freezing at -18--22℃ for 1-3h, and finally freezing at -75--85℃ overnight to obtain a frozen large-mouth bass muscle cell line, which can be stored in liquid nitrogen;
[0068] The method of freezing the large-mouth bass muscle cell line in step (2) preferably comprises first freezing at 4℃ for 30min, then freezing at -20℃ for 2h, and finally freezing at -80℃ overnight;
[0069] The culture solution in step (1) is the culture solution described above;
[0070] The muscle cell line of the largemouth bass in step (2) is the muscle cell line of the largemouth bass.
[0071] In the present application, the volume ratio of the culture solution mixed with the freezing medium in step (1) is 1-2:1-2, preferably 1:1.
[0072] In the present application, the freezing medium is composed of M199 medium, fetal bovine serum and dimethyl sulfoxide;
[0073] The volume ratio of the M199 medium, fetal bovine serum and dimethyl sulfoxide is 2-4:1:1, preferably 3:1:1.
[0074] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.
[0075] DMEM high sugar, DMEM / F12, L15 medium, M199 medium, cell culture medium, 100x double-antibiotic concentrate (containing 10000 IU / mL penicillin, 10000 μg / mL streptomycin), sodium pyruvate in the present application are purchased from Thermo Fisher Scientific (China) Co., Ltd., and each medium is prepared according to the product instructions of the company. Hydroxyethylpiperazine ethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO) are purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Trypsin is purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd.
[0076] Preparation of PBS buffer solution: take 8g of NaCl, 0.2g of KCl, 3.58g of Na2HPO4·12H2O, and 0.27g of KH2PO4, and make up to 1L, sterilize at 121℃ for 20min, and store at 4℃.
[0077] Preparation of 1x PBS buffer solution: mix 50ml of PBS buffer solution with 1mL of 100x double-antibiotic concentrate evenly.
[0078] Preparation of PBS buffer solution with a double-antibiotic concentration of 1vt%: mix 1L of PBS buffer solution with 10mL of 100x double-antibiotic concentrate evenly.
[0079] Preparation of PBS buffer solution with a double-antibiotic concentration of 0.5vt%: mix 1L of PBS buffer solution with 5mL of 100x double-antibiotic concentrate evenly.
[0080] Preparation of PBS buffer solution with a double-antibiotic concentration of 0.25vt%: mix 1L of PBS buffer solution with 2.5mL of 100x double-antibiotic concentrate evenly.
[0081] Preparation of digestive juice: 2.5 g of porcine trypsin powder was dissolved in 1 L of sterile PBS buffer solution, 0.02% EDTA was added, filtered through a 0.22 μm filter membrane, aliquoted, and stored at 4°C for standby use, pH 7.3.
[0082] Example 1: Effect of different culture media and FBS concentrations on growth of largemouth bass muscle cells
[0083] 1. Acquisition of primary cells
[0084] (1) Acquisition of largemouth bass muscle: healthy and active largemouth bass were selected, placed on ice for 5 min to reduce fish activity, rinsed with 1x PBS buffer solution, wiped with paper towel to remove body surface mucus and water droplets, 75% alcohol was used to disinfect the body surface and wipe dry, and dissection was performed in a clean bench. The sterile scissors and forceps were used to carefully remove the largemouth bass muscle, which was sequentially soaked in PBS buffer solution with double-antibody concentrations of 1vt%, 0.5vt%, 0.25vt%, and 0vt%, each for 25 s, then soaked in 75% alcohol for 25 s, and washed with PBS buffer solution to remove residual alcohol, obtaining sterile largemouth bass muscle.
[0085] (2) Isolation of primary cells: the sterile largemouth bass muscle was cut into 1 mm 3 muscle pieces with sterile scissors and forceps, the freshly cut surface was exposed, and the muscle pieces were dispersed and placed in culture plates using a sterile gun head or forceps. The bottom of the tissue pieces was aspirated using a pipette, and the plates were inverted and dried for 2 h. Then, the tissue pieces were transferred to new culture plates, a small amount of cell culture medium was added (to infiltrate the tissue pieces but not to float them), and the plates were incubated at 28°C in an incubator overnight to obtain adherent primary cells. The adherent primary cells were digested with digestive juice to obtain discrete primary cells.
[0086] 2. Effect of different basal media on growth of largemouth bass muscle primary cells
[0087] To DMEM high-sugar, DMEM / F12 medium, M199 medium, and L15 medium, 1 mM of sodium pyruvate, 100 μg / mL of hydroxyethylpiperazine ethanesulfonic acid, 100 μmol / mL of β-mercaptoethanol, 100 IU / mL of penicillin, 100 μg / mL of streptomycin, and 20vt% of fetal bovine serum were added to obtain DMEM high-sugar medium, DMEM / F12 medium, M199 medium, and L15 medium, respectively.
[0088] Experimental scheme:
[0089] The number of primary cells obtained in "1. Acquisition of primary cells" was counted using a hemocytometer, and 1x10 4The primary cells of the Micropterus salmoides were inoculated in 24-well plates, and were cultured in DMEM high-sugar medium, DMEM / F12 medium, M199 medium and L15 medium respectively, the culture temperature was 28℃, and the cell counting was performed every day during the culture period by using a hemocytometer, each treatment group was repeated for 3 times, and the growth curves of the primary cells of the Micropterus salmoides in different basic culture media were drawn, and the results are shown in Figure 1 Fig. 1, in which the abscissa represents the culture days, and the ordinate represents the cell number.
[0090] According to Figure 1 It can be known that the primary cells of the Micropterus salmoides grow fastest in the M199 medium, grow second fastest in the L15 medium, and grow slowly in the DMEM high-sugar medium and the DMEM / F12 medium, so the optimal basic culture medium for the primary muscle cells of the Micropterus salmoides is the M199 medium.
[0091] 3. Effect of different fetal bovine serum concentrations on the growth of primary muscle cells of the Micropterus salmoides
[0092] The M199 medium was used as the basic culture medium, 1 mM of sodium pyruvate, 100 μg / mL of hydroxyethylpiperazine ethanesulfonic acid, 100 μmol / mL of β-mercaptoethanol, 100 IU / mL of penicillin, 100 μg / mL of streptomycin and 20 vt% of fetal bovine serum were added to obtain a culture medium with 20 vt% of fetal bovine serum, the concentration of the fetal bovine serum was adjusted to obtain culture media with 5 vt%, 10 vt% and 15 vt% of fetal bovine serum respectively.
[0093] Experimental scheme:
[0094] The number of the primary cells obtained in the "1. Acquisition of primary cells" was counted by using a hemocytometer, 1×10 4 The primary cells of the Micropterus salmoides were inoculated in 24-well plates, and were cultured in culture media with 5 vt%, 10 vt%, 15 vt% and 20 vt% of fetal bovine serum respectively, the culture temperature was 28℃, and the cell counting was performed every day during the culture period by using a hemocytometer, each treatment group was repeated for 3 times, and the growth curves of the primary cells of the Micropterus salmoides in different fetal bovine serum concentrations were drawn, and the results are shown in Figure 2 Fig. 2, in which the abscissa represents the culture days, and the ordinate represents the cell number.
[0095] According to Figure 2It was found that the growth rate of primary muscle cells from largemouth bass was directly proportional to the concentration of added fetal bovine serum (FBS). When the concentration of FBS in the culture medium was 5–10 vt%, the proliferation of primary muscle cells from largemouth bass was significantly slowed down. A FBS concentration of 5–10 vt significantly stimulated the proliferation of primary muscle cells from largemouth bass. A culture medium with a FBS concentration of 20 vt was slightly better than one with 15 vt. To ensure the stability of primary cell culture, the concentration of FBS in the culture medium was therefore 20 vt.
[0096] In summary, the optimal culture medium for the growth of primary muscle cells from largemouth bass is an M199 medium-based medium containing the following components at the following concentrations:
[0097] 1 mM sodium pyruvate, 100 μg / mL hydroxyethylpiperazine ethanesulfonic acid, 100 μmol / mL β-mercaptoethanol, 100 IU / mL penicillin, 100 μg / mL streptomycin, and 20 vt% fetal bovine serum.
[0098] Example 2: Method for establishing a muscle cell line of largemouth bass
[0099] (1) Obtaining largemouth bass muscle: Select healthy and vigorous largemouth bass, place them on ice for 5 minutes to reduce their vitality, rinse the surface with 1×PBS buffer solution, wipe off the mucus and water droplets on the body surface with paper towels, disinfect the body surface with 75% alcohol and dry it, and dissect it in a clean bench. Carefully remove the largemouth bass muscle with sterile scissors and forceps, and soak it in PBS buffer solution with antibiotic concentrations of 1vt%, 0.5vt%, 0.25vt%, and 0vt% in sequence for 25 seconds each time. Then soak it in 75% alcohol for 25 seconds. Use PBS buffer solution to wash away the residual alcohol to obtain sterile largemouth bass muscle.
[0100] (2) Primary cell isolation and culture: Using sterile scissors and forceps, sterile largemouth bass muscle was cut into 1mm pieces. 3 Muscle fragments were prepared by exposing fresh cut surfaces. Using a sterile pipette tip or forceps, the fragments were dispersed in a culture plate. Water was removed from the bottom of the tissue blocks using a pipette. The fragments were then inverted and air-dried for 2 hours before being transferred to a new culture plate. The optimal culture medium selected in Example 1 (enough to wet the tissue blocks without causing them to float) was added, and the plates were incubated at 28°C. Primary cells were isolated. The isolated primary cells are shown below. Figure 3 As shown, the culture medium was changed every two days to obtain adherent primary cells. The adherent primary cells are shown in the image. Figure 4 As shown.
[0101] (3) Subculture: After 8 days of culture, the primary cells achieved 100% adherence. Figure 5As shown, the primary culture solution was aspirated, and the cells were washed once with the digestion solution. Then, the cells were digested with the digestion solution until 90% of the cells were round and bright. After the digestion solution was carefully removed, the cells were dispersed by incubation for 30 seconds. The dispersed primary cells were mixed with the optimal culture solution screened in Example 1, and the mixture was subcultured at 28°C. The culture solution was replaced every 2 days to obtain a muscle cell line of Micropterus salmoides.
[0102] The obtained muscle cell line of Micropterus salmoides was subcultured at a ratio of 1:1. The muscle cell line of Micropterus salmoides at passage 15 is shown in Figure 6 The muscle cell line of Micropterus salmoides at passage 30 is shown in Figure 7 The muscle cell line of Micropterus salmoides at passage 45 is shown in Figure 8 The muscle cell line of Micropterus salmoides at passage 60 is shown in Figure 9
[0103] Example 3: Method for cryopreservation of the muscle cell line of Micropterus salmoides
[0104] Preparation of the cryopreservation medium: M199 medium, fetal bovine serum, and dimethyl sulfoxide were mixed at a ratio of 3:1:1 by volume to obtain the cryopreservation medium.
[0105] The method for cryopreservation of the muscle cell line of Micropterus salmoides comprises the following steps:
[0106] (1) The optimal culture solution screened in Example 1 was mixed with the cryopreservation medium at a ratio of 1:1 by volume to obtain a mixed medium.
[0107] (2) The muscle cell line of Micropterus salmoides at passage 60 in Example 2 was mixed with the mixed medium. The mixture was first frozen at 4°C for 30 minutes, then frozen at -20°C for 2 hours, and finally frozen at -80°C overnight to obtain the frozen muscle cell line of Micropterus salmoides, which was stored in liquid nitrogen to obtain the cryopreserved muscle cell line of Micropterus salmoides.
[0108] Example 4: Determination of the recovery ability of the cryopreserved muscle cell line of Micropterus salmoides
[0109] The muscle cell line of Micropterus salmoides cryopreserved in Example 3 was stored for 30 days. According to the principle of rapid thawing, the cryopreserved muscle cell line of Micropterus salmoides was rapidly thawed in a 28°C constant-temperature water bath. After confirming that there was no ice crystal residue in the cryopreservation tube, 5 times the volume of the optimal culture solution screened in Example 1 was added. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was resuspended with the optimal culture solution screened in Example 1. After mixing, the mixture was transferred into a culture bottle for further subculture. After 8 days of culture, the precipitate was obtained by centrifugation, resuspended with the optimal culture solution screened in Example 1, and obtained as a bacterial suspension. The bacterial suspension was stained with 0.4% trypan blue for 5 minutes, and the survival rate of the cells was determined by counting the number of dead and live cells using a hemocytometer.
[0110] The results show that the recovery rate of the frozen 30-day large mouth bass muscle cell line is 80%, the survival cells grow and divide normally, and the cell morphology and proliferation capacity have no significant difference compared with before freezing. To ensure high recovery rate, it is recommended to freeze the cell line once every 5 generations for backup, the more the number of passages, the better the cell quality, and the higher the success rate of recovery.
[0111] Example 5: Verification of the source of large mouth bass muscle cell line
[0112] 1. Cell and tissue RNA extraction
[0113] The large mouth bass muscle cell line constructed in Example 2 was subcultured for 20 times to obtain the 20th generation of large mouth bass muscle cell line. 1 mL of Trizol lysis solution was added, and the cells were incubated at room temperature for 10 min to fully lyse the cells. The lysate was obtained, and 0.2 mL of chloroform was added after incubation on ice for 5 min. The mixture was vortexed and incubated on ice for 4 min. The mixture was centrifuged at 12000g for 15 min, and the supernatant was collected. An equal volume of isopropanol was added, mixed well, and incubated for 10 min. Then the mixture was centrifuged at 12000g for 15 min to obtain the RNA precipitate. The RNA precipitate was washed twice with 75% ethanol (DEPC water) and dissolved in 20 μL of DNA / RNA free ddH2O to obtain the RNA of the large mouth bass muscle cell line.
[0114] Fresh muscle tissue was ground after quick freezing in liquid nitrogen, and 100 mg was added to a 1.5 ml centrifuge tube. 1 mL of Trizol lysis solution was added, and the mixture was incubated on ice for 5 min. Then 0.2 mL of chloroform was added, and the mixture was vortexed and incubated on ice for 4 min. The mixture was centrifuged at 12000g for 15 min, and the supernatant was collected. An equal volume of isopropanol was added, mixed well, and incubated for 10 min. Then the mixture was centrifuged at 12000g for 15 min to obtain the RNA precipitate. The RNA precipitate was washed twice with 75% ethanol (DEPC water) and dissolved in 20 μL of DNA / RNA free ddH2O to obtain the RNA of the large mouth bass muscle tissue.
[0115] 2. Reverse transcription and PCR amplification
[0116] The RNA of the large mouth bass muscle cell line and the RNA of the large mouth bass muscle tissue were used as templates, respectively, and were reverse transcribed into first-strand cDNA by Takara's reverse transcription kit. The specific gene 18S of the large mouth bass was downloaded from the NCBI database, and the primers were designed according to the gene 18S. The above cDNA was used as a template, and the designed primers were used to amplify the PCR fragments of the large mouth bass muscle cell line and the large mouth bass muscle tissue, respectively.
[0117] 3. Electrophoresis verification
[0118] The PCR amplified fragments of the muscle cell line of Micropterus salmoides and the PCR amplified fragments of the muscle tissue of Micropterus salmoides were subjected to gel electrophoresis, and the results are shown in Figure 10, wherein M represents a DL2000 marker band, 1 represents the muscle cell line of Micropterus salmoides, 2 represents ddH2O, 3 represents the muscle tissue of Micropterus salmoides, and 4 represents ddH2O.
[0119] According to the above embodiment, it can be known that the PCR amplified fragments of the muscle cell line of Micropterus salmoides are consistent with the PCR amplified fragments of the muscle tissue of Micropterus salmoides, which indicates that the muscle cell line of Micropterus salmoides does not mutate after being subcultured for many times and has good stability. Figure 10 According to the above embodiment, it can be known that the PCR amplified fragments of the muscle cell line of Micropterus salmoides are consistent with the PCR amplified fragments of the muscle tissue of Micropterus salmoides, which indicates that the muscle cell line of Micropterus salmoides does not mutate after being subcultured for many times and has good stability.
[0120] The culture solution used in the present application is a M199 medium-based culture solution, to which sodium pyruvate, hydroxyethylpiperazine ethanesulfonic acid, beta-mercaptoethanol, penicillin, streptomycin and fetal bovine serum are added, and the concentrations of the components are reasonably configured, so that the culture solution provides optimal conditions for the culture of the muscle cells of Micropterus salmoides and reduces the culture cost. Meanwhile, the culture solution provides a method for establishing the muscle cell line of Micropterus salmoides, which is simple and easy to operate, has high repeatability, and has good stability, can be subcultured continuously and can be stored at ultra-low temperature. The main cell morphology is irregular polygonal and fibrocyte-like, the cell division speed is fast, the subculture time is short, and the adhesion rate is high. The primary cell culture of Micropterus salmoides in the method takes a short time, and a large number of cells with good activity are obtained, which can be subcultured only after 8 days.
[0121] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.
Claims
1. A culture solution, characterized by, The culture solution is a M199 culture medium-based culture solution, and further comprises the following components at the following concentrations: 0.5-1.5 mM sodium pyruvate, 90-110 μg / mL hydroxyethylpiperazine ethanesulfonic acid, 90-110 μmol / mL β-mercaptoethanol, 90-110 IU / mL penicillin, 90-110 μg / mL streptomycin, and 15-20 vt% fetal bovine serum; and the pH value of the culture solution is 7.2-7.
4.
2. The culture solution of claim 1 is used in primary and / or subculture of a muscle cell line of Micropterus salmoides.
3. A method of establishing a muscle cell line of Micropterus salmoides, characterized in that, The method comprises the following steps: (1) obtaining muscle of Micropterus salmoides: washing and sterilizing the muscle of Micropterus salmoides to obtain sterile muscle of Micropterus salmoides; (2) isolating and culturing primary cells: cutting the sterile muscle of Micropterus salmoides to obtain muscle fragments, mixing the muscle fragments with the culture solution, and culturing at 26-30°C, and replacing the culture solution every 2-3 days to obtain adherent primary cells; (3) subculture: when the adherent rate of the adherent primary cells is greater than 80%, digesting the adherent primary cells with a digestion solution to obtain discrete primary cells, mixing the discrete primary cells with the culture solution for subculture, and obtaining a muscle cell line of Micropterus salmoides; The culture solution in steps (2) and (3) is the culture solution of claim 1.
4. The establishment method according to claim 3, characterized by, The washing method in step (1) is soaking the muscle of Micropterus salmoides in a PBS buffer containing double antibiotics for 3-5 times, each time for 15-30 s; The concentration of the double antibiotics in the PBS buffer is 0-1 vt%; The double antibiotics are penicillin and streptomycin; The concentration of penicillin in the double antibiotics is 9000-11000 IU / mL, and the concentration of streptomycin is 9000-11000 μg / mL; The sterilization method is soaking in 70-80% alcohol for 15-30 s.
5. The establishment method according to claim 3, characterized by, The volume of the muscle pieces in step (2) is 0.5-1.5 mm 3 ; the temperature of the culture in step (2) is 26-30°C; the digestive solution in step (3) is a trypsin solution; the concentration of the trypsin solution is 0.2-0.3 wt%.
6. The muscle cell line of Micropterus salmoides obtained by the establishment method of any one of claims 3-5.
7. The muscle cell line of Micropterus salmoides of claim 6 is used in the study of nutritional metabolism and immune regulation of Micropterus salmoides.
8. A method of cryopreservation of a large mouth bass muscle cell line, characterized by, The method comprises the following steps: (1) mixing the culture solution with a cryopreservation medium to obtain a mixed culture medium; (2) mixing the muscle cell line of Micropterus salmoides with the mixed culture medium, freezing at 3-5°C for 25-35 min first, then freezing at -18 to -22°C for 1-3 h, and finally freezing at -75 to -85°C overnight to obtain a frozen muscle cell line of Micropterus salmoides, which can be stored in liquid nitrogen; The culture solution in step (1) is the culture solution of claim 1. The muscle cell line of Micropterus salmoides in step (2) is the muscle cell line of Micropterus salmoides of claim 6.
9. The cryopreservation method of claim 8, wherein, The volume ratio of the culture solution to the cryopreservation medium in step (1) is 1-2:1-2.
10. The cryopreservation method of claim 9, wherein, The cryopreservation medium is composed of M199 culture medium, fetal bovine serum, and dimethyl sulfoxide; The volume ratio of the M199 culture medium, fetal bovine serum, and dimethyl sulfoxide is 2-4:1:1.