Culture medium and culture method for fish primary liver cells

Abstract

The invention belongs to the technical field of cell biology, and particularly discloses a culture medium and a culture method of fish primary liver cells. The culture medium of the fish primary liver cells comprises the following components: 90% of an M199 culture medium, 10% of fetal calf serum, 100 U / mL of penicillin, 0.1 mg / mL of streptomycin, 5 ng / mL of EGF, 10 [mu] U / ml of insulin and 10 mM of dexamethasone. Based on the culture medium, an efficient and stable fish liver cell culture method is developed by combining a standardized anesthesia and disinfection process, a precise dissection and liver sampling method, an optimized tissue digestion and cell separation scheme, an efficient cell purification and adherent culture system, a stable passage technology and the like; a standardized hepatocyte line establishment operation process is defined, and the problems that species binding exists in an existing fish liver cell culture method, and a standardized culture process suitable for multiple fish fingerlings is difficult to form are solved.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, specifically to a culture medium and culture method for primary fish liver cells. Background Technology

[0002] The fish liver consists of a very thin serous membrane, hepatocytes, and the sinusoids and bile canaliculi between them. Hepatocytes play a crucial role in the liver's metabolism and detoxification processes. Therefore, clarifying the gene expression and environmental responses of fish liver cells is helpful for further understanding the metabolic adaptation and toxin tolerance processes in fish. Current primary-passage culture methods for fish liver cells commonly use collagenase digestion, while passage culture often employs trypsin digestion. Current primary-passage culture methods for fish liver cells are often species-specific (step parameters are based on specific fish species), making it difficult to develop standardized culture procedures applicable to multiple fish species. Summary of the Invention

[0003] To address the above problems, this invention provides a standardized and quantifiable method for culturing fish liver cells.

[0004] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows (the "%" in the following technical solution refers to volume fraction):

[0005] In a first aspect, the present invention provides a culture medium for primary fish liver cells, comprising the following components: M199 medium, fetal bovine serum, penicillin, streptomycin, epidermal growth factor (EGF), insulin, and dexamethasone.

[0006] Preferably, the components of the culture medium for the primary fish liver cells are as follows: 90% M199 medium, 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 5 ng / mL EGF, 10 μU / mL insulin, and 10 mM dexamethasone. The concentrations of penicillin, streptomycin, EGF, insulin, and dexamethasone in this formulation are calculated based on the sum of the volumes of M199 medium and fetal bovine serum.

[0007] Secondly, the present invention provides a method for culturing primary fish liver cells, comprising the following steps:

[0008] S1. Anesthetize and disinfect the fish, then cut open the fish to remove the liver tissue, cut it into small pieces, and wash it.

[0009] S2. Mix the washed liver tissue with digestive juice for digestion, and gently shake in a shaker to separate the liver cells in the tissue;

[0010] S3. Add M199 medium containing fetal bovine serum and two antibiotics (penicillin-streptomycin) to terminate digestion, centrifuge, discard the supernatant and resuspend in M199 medium;

[0011] S4. Wash the dispersed cell suspension with M199 medium containing fetal bovine serum and two antibiotics, then filter it with a cell sieve to resuspend the cells in the above liver cell medium that does not contain phenol red (phenol red has certain estrogenic activity, and fish liver cells are sensitive to estrogen).

[0012] S5. Seed the cells suspended in the culture medium into 24-well plates and incubate them in a cell culture incubator at 28°C and 5% CO2 for three days;

[0013] S6. Three days later, remove the 24-well plate, aspirate the non-adherent hepatocytes and culture medium, add M199 cell culture medium containing fetal bovine serum and two antibiotics, and then incubate at 28°C and 5% CO2 for three days.

[0014] S7. Three days later, remove the 24-well plate, aspirate the culture medium, wash the adherent cells and add trypsin to digest them. During the digestion process, gently tap the culture dish to make most of the adherent cells float.

[0015] S8. Add M199 medium containing fetal bovine serum to terminate digestion, centrifuge and resuspend;

[0016] S9. Filter the cell suspension using a cell sieve, then seed it into a 24-well plate and incubate at 28°C and 5% CO2 for 24 hours until it adheres to the plate and grows.

[0017] Preferably, in step S1, sexually mature male fish are taken and anesthetized with 2.5 mg / L of ethyl 3-aminobenzoate methanesulfonate (MS-222) anesthetic in the water in which they are located. After the fish are deeply anesthetized, they are removed with forceps, and the surface of the fish is disinfected by spraying with 75% alcohol. The anesthesia and disinfection in this step aims to standardize the physiological state of the cell source, reduce the interference of individual differences on the culture results, and is the basis for improving the reproducibility of the method.

[0018] Preferably, in step S1, the method of cutting open the fish body is as follows: In a clean bench, using sterilized dissecting scissors, cut the skin and muscle tissue along the midline of the abdomen of the male fish to be sampled, moving forward from the ventral opening (tilting the scissors upward at about 15° to avoid damaging the internal organs), until below the base of the pectoral fin. Then, turn the cut abdominal wall tissue toward the ventral side of the fish body to fully expose the abdominal viscera.

[0019] Preferably, in step S1, the liver tissue is taken as follows: liver tissue (generally about 5g; if the fish is small, the amount can be reduced or the whole liver can be taken) is taken using sterilized tweezers and placed in a culture dish containing L-15 culture medium preheated to 28℃. During the process, other tissues should be avoided.

[0020] Preferably, in step S1, the washing is performed three times with 60% L-15 culture medium, followed by three times with 1×PBS solution.

[0021] Preferably, in step S2, the digestive solution is an EDTA solution containing 0.25% trypsin, the digestion temperature is 28°C, and the digestion is performed by gently shaking at 800 rpm for 5 minutes in a shaker. Gently shaking at 800 rpm for 5 minutes, compared to conventional static digestion or vigorous shaking, achieves a balance between liver cell yield and activity, and is particularly suitable for fish liver cells with fragile cell walls.

[0022] Preferably, in step S3, the centrifugation conditions are 800g for 5 minutes at 28°C, the supernatant is discarded and the mixture is resuspended in M199 medium.

[0023] Preferably, in step S4, the dispersed cell suspension is washed three times with M199 medium and then filtered through a 70 μm cell sieve to resuspend the cells in phenol red-free liver cell culture medium.

[0024] Preferably, in step S5, 5 × 10⁵ seeds are inoculated into each well of the 24-well plate. 5 Each cell.

[0025] Preferably, in step S7, adherent cells are washed with 10 mL of PBS solution and repeated three times, and 2 mL of 0.25% trypsin is added for digestion for 8 min.

[0026] Preferably, in step S8, the cells are centrifuged at 400g for 3 minutes, the supernatant is discarded, and this process is repeated 3 times; 15mL of cell culture medium is added to resuspend the cells, and the cells are counted. The number of viable cells per milliliter should exceed 1×10⁻⁶. 6 Each cell.

[0027] Preferably, in step S9, the cell suspension is filtered using a cell sieve with a pore size of 70 μm, the filtered cell suspension is collected, and seeded into a 24-well plate, with 500 μL seeded into each well. The above-described method for culturing primary fish liver cells of the present invention has the following key points:

[0028] 1. Standardized anesthesia and disinfection procedures: MS-222 anesthetic at a concentration of 2.5 mg / L is used to ensure deep anesthesia of the fish and reduce stress damage. Surface disinfection with 75% alcohol is also used to reduce the risk of microbial contamination.

[0029] 2. Precise dissection and liver sampling method: Cut along the midline of the abdomen (scissors tilted upwards) to avoid damaging internal organs or cutting. The liver tissue is quickly separated, minimizing interference from other tissues.

[0030] 3. Optimized tissue digestion and cell separation protocol: Add 0.25% trypsin-EDTA and digest at 800 rpm for 5 min to improve single cell yield. Digestion is terminated with M199 medium containing 10% fetal bovine serum to protect cell viability.

[0031] 4. Innovative Liver Cell Culture Medium Formula: 90% M199 medium, 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 5 ng / mL epidermal growth factor (EGF), 10 μU / mL insulin, and 10 mM dexamethasone. This liver cell culture medium formula is an optimization of the classic M199 medium. Fetal bovine serum provides abundant growth factors necessary for cell proliferation, penicillin and streptomycin inhibit bacterial proliferation, EGF promotes cell proliferation, insulin regulates cell metabolism, and dexamethasone strengthens intercellular connections. These components work synergistically to ensure liver cell survival, proliferation, and functional maintenance, making it suitable for primary liver cell culture.

[0032] 5. A highly efficient cell purification and adherent culture system was developed: Considering the size of hepatocytes, a 70μm cell sieve was used to filter and remove undigested tissue clumps. A two-stage culture method was also used (first 3 days for adhesion screening, then 3 days for amplification) to improve hepatocyte purity.

[0033] 6. Stable passage technique: After digesting with 0.25% trypsin for 8 minutes, gently tap the culture dish to gently detach the adherent cells. Centrifuge at 400g for 3 minutes and filter through a 70μm cell sieve to ensure the homogeneity of the passaged cell suspension.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This invention develops a highly efficient and stable fish hepatocyte culture medium formula and supporting culture system, clarifies the standardized hepatocyte establishment operation procedure, and is applicable to primary-passage culture of various fish (such as zebrafish, medaka, etc.). It provides a reliable in vitro research model for in-depth research on key physiological processes of fish hepatocytes such as metabolism, detoxification mechanism and environmental adaptability, and lays the foundation for promoting aquatic animal physiological research and toxicological assessment. Attached Figure Description

[0036] Figure 1 Microscopic view of zebrafish liver cells cultured in vitro for three days in Example 1.

[0037] Figure 2Microscopic view of zebrafish liver cells cultured in vitro for six days in Example 1.

[0038] Figure 3 Microscopic view of zebrafish liver cells after passage in Example 1.

[0039] Figure 4 Microscopic view of medaka liver cells cultured in vitro for three days in Example 2.

[0040] Figure 5 Microscopic view of medaka liver cells cultured in vitro for six days in Example 2.

[0041] Figure 6 Microscopic view of medaka liver cells after passage in Example 2.

[0042] Figure 1-6 The two images on the left and right are field-of-view images from different positions under a microscope. Detailed Implementation

[0043] This invention constructs a standardized and quantifiable method for culturing fish liver cells through a series of synergistically optimized key technical features, specifically including the following steps:

[0044] (1) Take sexually mature male fish and anesthetize them with 2.5 mg / L of ethyl 3-aminobenzoate methanesulfonate (MS-222) in the water. After the fish is deeply anesthetized, remove it with forceps and spray the surface of the fish with 75% alcohol for disinfection. Note: MS-222 must be administered at a precise concentration (2.5 mg / L) to ensure deep anesthesia and complete relaxation of the fish, avoiding stress reactions that could interfere with dissection. Note: 75% alcohol should be applied by surface spraying rather than soaking to avoid tissue penetration damage and to kill surface microorganisms.

[0045] (2) In a clean bench, use sterilized dissecting scissors to cut the skin and muscle tissue along the midline of the abdomen of the male fish to be sampled, forward from the ventral opening (tilt the scissors slightly upward to avoid damaging the internal organs, causing leakage of digestive enzymes or even cytotoxicity), until below the base of the pectoral fin. Then turn the cut abdominal wall tissue toward the ventral side of the fish to fully expose the abdominal viscera.

[0046] (3) Use sterilized forceps to take liver tissue (generally about 5g; if the fish is small, the amount can be reduced or the whole liver can be taken), and place it in a culture dish containing L-15 medium preheated to 28℃. Avoid touching other tissues during the process. The 28℃ L-15 medium can maintain the stability of cell metabolism and avoid damage to cell activity caused by cold shock.

[0047] (4) Gently cut the liver tissue into small pieces with a scalpel, then transfer them to 50 mL centrifuge tubes, wash three times with 60% L-15 medium, and then wash three times with 1×PBS solution. Specifically, wash with L-15 (nutrient maintenance) first and then with PBS (thorough cleaning) to remove blood, residual body fluids and other impurities step by step.

[0048] (5) The washed liver tissue was mixed with 0.25% trypsin-EDTA solution at 28°C for digestion, and then gently shaken at 800 rpm for 5 minutes in a shaker to separate the hepatocytes in the tissue. Among them, the gentle shaking at 800 rpm can balance the dissociation efficiency and cell protection; the low temperature digestion at 28°C (not 37°C) is suitable for the characteristics of fish cells.

[0049] (6) Subsequently, add an equal volume of M199 medium containing 10% fetal bovine serum and two antibiotics (penicillin-streptomycin) to the entire mixture to terminate the digestion process. Then, centrifuge again at 800g for 5min at 28°C. After centrifugation, discard the supernatant and resuspend in M199 medium.

[0050] (7) The dispersed cell suspension was washed three times with M199 medium containing 10% fetal bovine serum and two antibiotics, then filtered through a 70 μm cell sieve. The filtrate (single-cell suspension) was centrifuged, the supernatant was discarded, and liver cell culture medium was added to resuspend the cells in phenol red-free liver cell culture medium. The liver cell culture medium formula was: 90% M199 medium, 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 5 ng / mL epidermal growth factor (EGF), 10 μU / mL insulin, and 10 mM dexamethasone. The 70 μm cell sieve filtration removes undigested tissue fragments, ensuring a single-cell suspension and preventing cell aggregation that could affect cell adhesion uniformity. The liver cell culture medium used in this invention is phenol red-free. This seemingly minor change effectively avoids background interference from culture medium components on liver cell metabolism and gene expression, which is particularly important for toxicological studies involving endocrine disruptors, a point easily overlooked in conventional culture.

[0051] (8) Seed the cells suspended in the culture medium into 24-well plates (5 × 10⁶ cells per well). 5 The cells were cultured in a cell culture incubator at 28°C and 5% CO2 for three days. Note that: insufficient inoculation density leads to insufficient paracrine signaling and decreased cell adhesion; 28°C is suitable for fish cell culture and can optimize metabolism.

[0052] (9) After three days, take out the 24-well plate, observe the adhesion of hepatocytes, and place it in a clean bench to remove the non-adherent hepatocytes and culture medium to avoid the release of toxic substances by dead cells. Then add 30 mL of the above M199 cell culture medium containing 10% fetal bovine serum and two antibiotics to the 24-well plate and place it in a clean bench at 28°C and 5% carbon dioxide for three days.

[0053] (10) After three days, remove the cell culture dish and observe the adhesion of hepatocytes. At this time, the percentage of hepatocytes adhering to the wall can be observed to be 60-70%. Place the cell culture dish on a clean bench, aspirate the culture medium, add 10 mL of PBS solution to wash the adherent cells and repeat three times, and add 2 mL of 0.25% trypsin to digest for 8 min. During digestion, gently tap the culture dish to make most of the adherent cells float.

[0054] (11) Add 30 mL of M199 medium containing 10% fetal bovine serum to stop digestion, and transfer the cells to a 50 mL centrifuge tube using a sterile Pasteur dropper. Centrifuge at 400 g for 3 min, discard the supernatant, and repeat 3 times.

[0055] (12) Add 15 mL of M199 medium containing 10% fetal bovine serum, resuspend the cells, and count the cells. The number of viable cells per milliliter should exceed 1 × 10⁻⁶. 6 Each cell.

[0056] (13) The cell suspension was filtered using a cell sieve with a pore size of 70 μm. The filtered cell suspension was collected and seeded into a 24-well plate with 500 μL per well. The plate was then placed in a 5% CO2, 28°C cell culture incubator for 24 hours to allow the cells to adhere and grow.

[0057] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0058] Example 1: Primary and passaged culture of zebrafish hepatocytes

[0059] This embodiment provides an application example of the above-described fish liver cell culture method in zebrafish. The culture process of primary zebrafish liver cells is as follows:

[0060] (1) Take a male zebrafish that has reached sexual maturity and anesthetize it by injecting 2.5 mg / L of ethyl 3-aminobenzoate methanesulfonate (MS-222) anesthetic into the water where it is located. After the fish is deeply anesthetized, take it out with tweezers and spray the surface of the fish with 75% alcohol for disinfection.

[0061] (2) In a clean bench, use sterilized dissecting scissors to cut the skin and muscle tissue along the midline of the abdomen of the male zebrafish to be sampled (tilt the scissors slightly upward to avoid damaging the internal organs) until below the base of the pectoral fin. Then turn the cut abdominal wall tissue toward the ventral side of the fish to fully expose the abdominal viscera.

[0062] (3) Use sterilized tweezers to take about 1g of liver tissue and place it in a culture dish containing L-15 medium preheated to 28℃. Avoid touching other tissues during the process.

[0063] (4) Use a scalpel to gently cut the liver tissue into small pieces, then transfer them to 50 mL centrifuge tubes, wash three times with 60% L-15 medium, and then wash three times with 1×PBS solution.

[0064] (5) The washed liver tissue was mixed with 0.25% trypsin-EDTA solution at 28°C for digestion, and then gently shaken in a shaker at 800 rpm for 5 minutes to separate the hepatocytes in the tissue.

[0065] (6) Then, add an equal volume of M199 medium containing 10% fetal bovine serum and two antibiotics (100 U / mL penicillin and 0.1 mg / mL streptomycin) to the whole mixture to terminate the digestion process. Then, centrifuge again at 800 g for 5 min at 28 °C, discard the supernatant, and resuspend the cells in M199 medium to obtain a cell suspension.

[0066] (7) Wash the dispersed cell suspension three times with M199 medium containing 10% fetal bovine serum and two antibiotics, then filter through a 70 μm cell sieve. Centrifuge the filtrate (single-cell suspension), discard the supernatant, and add liver cell culture medium to resuspend the cells in phenol red-free liver cell culture medium. Liver cell culture medium formula: 90% M199 medium, 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 5 ng / mL epidermal growth factor (EGF), 10 μU / mL insulin, 10 mM dexamethasone.

[0067] (8) Seed the cells suspended in the culture medium into 24-well plates (5 × 10⁶ cells per well). 5 The zebrafish liver cells were cultured in a cell culture incubator at 28°C and 5% carbon dioxide for three days (after three days, the zebrafish liver cells were as follows: ...). Figure 1 (As shown).

[0068] (9) Three days later, remove the 24-well plate, observe the hepatocyte adhesion, and place it in a clean bench to remove the non-adhered hepatocytes and culture medium. Add 30 mL of the above-mentioned M199 cell culture medium containing 10% fetal bovine serum and two antibiotics, and re-culture at 28°C and 5% carbon dioxide for three days (zebrafish hepatocytes after six days of culture are as follows). Figure 2 (As shown).

[0069] (10) After three days, remove the cell culture dish and observe the adhesion of liver cells. At this time, the percentage of liver cells adhering to the wall can be observed to be 60-70%. Place the cell culture dish on a clean bench, aspirate the culture medium, add 10 mL of PBS solution to wash the adherent cells and repeat three times, and add 2 mL of 0.25% trypsin to digest for 8 min. During digestion, gently tap the culture dish to make most of the adherent cells float.

[0070] (11) Add 30 mL of M199 medium containing 10% fetal bovine serum to stop digestion, and transfer the cells to a 50 mL centrifuge tube using a sterile Pasteur dropper. Centrifuge at 400 g for 3 min, discard the supernatant, and repeat 3 times.

[0071] (12) Add 15 mL of M199 medium containing 10% fetal bovine serum, resuspend the cells, and count the cells. The number of viable cells per milliliter should exceed 1 × 10⁻⁶. 6 Each cell.

[0072] (13) The cell suspension was filtered using a 70 μm pore size cell sieve. The filtered cell suspension was collected and seeded into 24-well plates at 500 μL per well. The plates were then incubated in a 5% CO2, 28°C cell culture incubator for 24 hours until the cells adhered and grew. The growth was as follows: Figure 3 As shown.

[0073] Depend on Figure 1-3 It can be seen that zebrafish liver cells adhered to the culture wall on the 3rd day of primary culture, and were polygonal with clear outlines; the number of cells increased on the 6th day, forming island-like clones; 24 hours after passage, the cells were evenly dispersed, with good morphology and adhesion rate, indicating that the zebrafish liver cell culture was successful.

[0074] Example 2: Primary and passaged culture of medaka hepatocytes

[0075] This embodiment provides an application example of the above-mentioned fish liver cell culture method in medaka. The culture process of primary medaka liver cells is as follows:

[0076] (1) Take a male medaka that has reached sexual maturity and anesthetize it with 2.5 mg / L of ethyl 3-aminobenzoate methanesulfonate (MS-222) anesthetic. After the fish is deeply anesthetized, take it out with tweezers and spray the surface of the fish with 75% alcohol for disinfection.

[0077] (2) In a clean bench, use sterilized dissecting scissors to cut along the vent of the male medaka to be sampled, along the midline of the abdomen, and cut open the skin and muscle tissue (tilt the scissors slightly upward to avoid damaging the internal organs) until below the base of the pectoral fin. Then turn the cut abdominal wall tissue toward the ventral side of the fish to fully expose the abdominal viscera.

[0078] (3) Use sterilized tweezers to take about 1g of liver tissue and place it in a culture dish containing L-15 medium preheated to 28℃. Avoid touching other tissues during the process.

[0079] (4) Use a scalpel to gently cut the liver tissue into small pieces, then transfer them to 50 mL centrifuge tubes, wash three times with 60% L-15 medium, and then wash three times with 1×PBS solution.

[0080] (5) The washed liver tissue was mixed with 0.25% trypsin-EDTA solution at 28°C for digestion, and then gently shaken in a shaker at 800 rpm for 5 minutes to separate the hepatocytes in the tissue.

[0081] (6) Then, add an equal volume of M199 medium containing 10% fetal bovine serum and two antibiotics (100 U / mL penicillin and 0.1 mg / mL streptomycin) to the whole mixture to terminate the digestion process. Then, centrifuge again at 800 g for 5 min at 28 °C, discard the supernatant, and resuspend the cells in M199 medium to obtain a cell suspension.

[0082] (7) Wash the dispersed cell suspension three times with M199 medium containing 10% fetal bovine serum and two antibiotics, then filter through a 70 μm cell sieve. Centrifuge the filtrate (single-cell suspension), discard the supernatant, and add liver cell culture medium to resuspend the cells in phenol red-free liver cell culture medium. Liver cell culture medium formula: 90% M199 medium, 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 5 ng / mL epidermal growth factor (EGF), 10 μU / mL insulin, 10 mM dexamethasone.

[0083] (8) Seed the cells suspended in the culture medium into 24-well plates (5 × 10⁶ cells per well). 5 The cells were cultured in a cell culture incubator at 28°C and 5% carbon dioxide for three days (after three days, the medaka liver cells were as follows). Figure 4 (As shown).

[0084] (9) Three days later, remove the 24-well plate, observe the adherence of hepatocytes, and place it in a clean bench to remove unadhered hepatocytes and culture medium. Add 30 mL of the above-mentioned M199 cell culture medium containing 10% fetal bovine serum and two antibiotics, and re-culture at 28°C and 5% carbon dioxide for three days (the medaka hepatocytes after six days of culture are as follows). Figure 5 (As shown).

[0085] (10) After three days, remove the cell culture dish and observe the adhesion of liver cells. At this time, the percentage of liver cells adhering to the wall can be observed to be 60-70%. Place the cell culture dish on a clean bench, aspirate the culture medium, add 10 mL of PBS solution to wash the adherent cells and repeat three times, and add 2 mL of 0.25% trypsin to digest for 8 min. During digestion, gently tap the culture dish to make most of the adherent cells float.

[0086] (11) Add 30 mL of M199 medium containing 10% fetal bovine serum to stop digestion, and transfer the cells to a 50 mL centrifuge tube using a sterile Pasteur dropper. Centrifuge at 400 g for 3 min, discard the supernatant, and repeat 3 times.

[0087] (12) Add 15 mL of M199 medium containing 10% fetal bovine serum, resuspend the cells, and count the cells. The number of viable cells per milliliter should exceed 1 × 10⁻⁶. 6 Each cell.

[0088] (13) The cell suspension was filtered using a 70 μm pore size cell sieve. The filtered cell suspension was collected and seeded into 24-well plates at 500 μL per well. The plates were then incubated in a 5% CO2, 28°C cell culture incubator for 24 hours until the cells adhered and grew. The growth was as follows: Figure 6 As shown.

[0089] Depend on Figure 4-6 It can be seen that the results of medaka hepatocyte culture are consistent with those of zebrafish. The cells adhere well on day 3, form a dense monolayer on day 6, and are evenly dispersed after passage, with good morphology and adhesion rate. This proves that the method of the present invention can successfully culture fish hepatocytes and is applicable to different fish species.

[0090] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims of the present invention, will be protected by patent law.

Claims

1. A culture medium for primary fish liver cells, characterized in that, It includes the following components: M199 medium, fetal bovine serum, penicillin, streptomycin, EGF, insulin, and dexamethasone.

2. The culture medium for primary fish liver cells according to claim 2, characterized in that, The contents of each component are as follows: 90% M199 medium, 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 5 ng / mL EGF, 10 μU / mL insulin, and 10 mM dexamethasone.

3. A method for culturing primary fish liver cells, characterized in that, Includes the following steps: S1. Anesthetize and disinfect the fish, then cut open the fish to remove the liver tissue, cut it into small pieces, and wash it. S2. Mix the washed liver tissue with digestive juice for digestion, and gently shake in a shaker to separate the liver cells in the tissue; S3. Add M199 medium containing fetal bovine serum and two antibiotics to stop digestion, centrifuge and resuspend; S4. The dispersed cell suspension is washed with M199 medium containing fetal bovine serum and two antibiotics, and then filtered with a cell sieve to resuspend the cells in a culture medium for primary fish liver cells as described in claim 1 or 2 that does not contain phenol red. S5. Seed the cells suspended in the culture medium into 24-well plates and incubate them in a cell culture incubator at 28°C and 5% CO2 for three days; S6. Three days later, remove the 24-well plate, aspirate the non-adherent hepatocytes and culture medium, add M199 cell culture medium containing fetal bovine serum and two antibiotics, and then incubate at 28°C and 5% CO2 for three days. S7. Three days later, remove the 24-well plate, aspirate the culture medium, wash the adherent cells and add trypsin to digest them. During the digestion process, gently tap the culture dish to make most of the adherent cells float. S8. Add M199 medium containing fetal bovine serum to terminate digestion, centrifuge and resuspend; S9. Filter the cell suspension using a cell sieve, then seed it into a 24-well plate and incubate at 28°C and 5% CO2 for 24 hours until it adheres to the plate and grows.

4. The method for culturing primary fish liver cells according to claim 3, characterized in that, In step S1, male fish that have reached sexual maturity are taken and anesthetized by adding ethyl 3-aminobenzoate methanesulfonate to the water in which they are located. After anesthesia, the surface of the fish is sprayed with 75% alcohol for disinfection.

5. The method for culturing primary fish liver cells according to claim 4, characterized in that, In step S1, after disinfection, use dissecting scissors to cut along the vent of the male fish to be sampled, tilting the scissors upwards and cutting through the skin and muscle tissue along the midline of the abdomen until below the base of the pectoral fin. Then, turn the cut abdominal wall tissue toward the ventral side of the fish to fully expose the visceral mass in the abdominal cavity. Then, liver tissue was removed with forceps, placed in a culture dish containing L-15 medium preheated to 28°C, and then cut into pieces.

6. The method for culturing primary fish liver cells according to claim 3, characterized in that, In step S2, the digestive solution is an EDTA solution containing 0.25% trypsin, the digestion temperature is 28°C, and the digestion is carried out by gently shaking at 800 rpm for 5 minutes in a shaker.

7. The method for culturing primary fish liver cells according to claim 3, characterized in that, In step S3, the centrifugation conditions are 28°C and centrifugation at 800g for 5 minutes.

8. The method for culturing primary fish liver cells according to claim 3, characterized in that, In step S7, adherent cells were washed with PBS solution and repeated three times, and then digested with 0.25% trypsin for 8 min.

9. The method for culturing primary fish liver cells according to claim 3, characterized in that, In step S8, centrifuge at 400g for 3 minutes, discard the supernatant, and repeat 3 times; resuspend the cells in 15mL of cell culture medium, count the cells, and the number of viable cells per milliliter should exceed 1×10⁻⁶. 6 Each cell.

10. The method for culturing primary fish liver cells according to claim 3, characterized in that, In steps S4 and S9, the cell sieve used has a size of 70 μm.

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