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CN-122012376-A - Method for establishing liver cell line of micropterus salmoides

CN122012376ACN 122012376 ACN122012376 ACN 122012376ACN-122012376-A

Abstract

The invention provides a method for establishing a liver cell line of micropterus salmoides, belonging to the technical field of cell culture. The invention adopts a tissue block method to separate primary cells of the liver of the micropterus salmoides from the liver tissue of the micropterus salmoides, and the primary cells of the liver of the micropterus salmoides are subjected to subculture to obtain a liver cell line of the micropterus salmoides. The invention establishes a stable and standard culture method of the liver cell line of the micropterus salmoides, which is simple and easy to operate, has strong repeatability of the construction method, good stability of the constructed cell line, vigorous cell division and short passage time. The method provided by the invention has important significance for developing in-vitro nutrition metabolism research, disease mechanism analysis, functional feed development and the like of the largehead jewfish, and can also provide key technical support for healthy cultivation of the largehead jewfish and quality improvement and synergy of industry.

Inventors

  • WANG QINGCHAO
  • REN HUIJUAN
  • ZOU JIAHONG
  • TANG WEIJUN
  • YUE YAOQIANG
  • XIONG KEXIN
  • LIU SHIHAO

Assignees

  • 华中农业大学

Dates

Publication Date
20260512
Application Date
20260211

Claims (10)

  1. 1. A culture medium for culturing a liver cell line of micropterus salmoides, which is characterized by comprising a primary culture medium; The primary culture medium is a DMEM/F-12 basic culture medium containing 18-22% fetal bovine serum, 4.5-5.5 mug/mL human FGF and 3.5-4.5% first antibiotic compound liquid; the first antibiotic compound liquid comprises 390-410U/mL penicillin, 0.38-0.42 mg/mL streptomycin and 180-220 mug/mL amphotericin.
  2. 2. The culture medium for the liver cell line culture of micropterus salmoides according to claim 1, wherein the primary culture medium is a DMEM/F-12 basal medium containing 20% fetal bovine serum, 5 mug/mL human FGF and 4% first antibiotic compound solution by volume concentration; The first antibiotic compound liquid comprises 400U/mL penicillin, 0.4. 0.4 mg/mL streptomycin and 200 mug/mL amphotericin.
  3. 3. The culture medium for the liver cell line culture of micropterus salmoides according to claim 1, further comprising a subculture medium; The subculture medium is a DMEM/F-12 basal medium containing 18-22% fetal bovine serum and 1.8-2.2% second antibiotic compound liquid in volume concentration; The second antibiotic compound liquid comprises 180-220U/mL penicillin, 0.18-0.22 mg/mL streptomycin and 90-110 mug/mL amphotericin.
  4. 4. The culture medium for the liver cell line of micropterus salmoides according to claim 3, wherein the subculture medium is a DMEM/F-12 basal medium containing the following concentration components, namely, 20% fetal bovine serum and 2% second antibiotic compound solution by volume concentration; the second antibiotic compound liquid comprises 200U/mL penicillin, 0.2 mg/mL streptomycin and 100 mug/mL amphotericin.
  5. 5. The culture medium for culturing the micropterus salmoides liver cell line according to any one of claims 1 to 4, further comprising a cell freezing solution; The cell freezing solution comprises fetal bovine serum and DMSO in a volume ratio of 8-12:0.8-1.2.
  6. 6. The method for establishing the liver cell line of the largemouth bass is characterized by comprising the following steps of: Primary culturing liver tissue fragments of micropterus salmoides in a primary culture medium in any one of the culture media of claims 1-5 to obtain a cell monolayer; after preparing single cells from the cell monolayer, placing the single cells in a subculture medium in any one of the culture mediums of claims 1-5 for subculture to obtain the liver cell line of the micropterus salmoides.
  7. 7. The method according to claim 6, wherein the primary culture is performed at a temperature of 27-29 ℃ for 10-15 days, wherein the primary culture is performed at a concentration of 5% CO 2 ; The primary culture is replaced once a day with primary culture medium.
  8. 8. The method according to claim 6, wherein the subculture is performed once every 2 days; The passage number of the subculture comprises 10-120 passages; and carrying out passage according to the ratio of 1:1 for 1 to 10 generations, and carrying out passage according to the ratio of 1:2 after 10 generations.
  9. 9. The method according to any one of claims 6 to 8, wherein the largemouth black bass liver cell line further comprises preservation in a cell preservation solution; the cell preservation solution according to claim 5.
  10. 10. The method of claim 9, wherein the largemouth bass liver cell line comprises cell resuscitation; The cell resuscitating comprises melting in a water bath, separating cells, and re-suspending in a subculture medium; The melting temperature of the water bath is 36-38 ℃ and the time is 50-70 s.

Description

Method for establishing liver cell line of micropterus salmoides Technical Field The application belongs to the technical field of cell culture, and particularly relates to a method for establishing a liver cell line of micropterus salmoides. Background The micropterus salmoides (Micropterus salmoides) are also called as micropterus salmoides, belonging to the order of micropterus salmoides, the family of acanthus, the genus of micropterus, and have the characteristics of fine and delicious meat, rich nutrition, no intramuscular thorns, rapid growth and strong adaptability, and are one of main characteristic freshwater aquaculture varieties in China. Liver metabolic diseases such as fatty liver and hepatomegaly are also easy to occur in the culture process of the largehead jewfish. The fish liver plays a central role in energy metabolism, nutrient substance conversion and storage, and the influence of an energy distribution mechanism, nutrition demand characteristics and feed composition on the growth performance and the health state can be deeply analyzed by researching the liver of the micropterus salmoides, so that scientific basis is provided for realizing accurate nutrition regulation and control and promoting the sustainable development of the aquaculture industry. The cell line is an important platform for developing research on physiological, nutritional, disease and genetic mechanisms of fish, the current method for establishing the liver cell line of the micropterus salmoides reports that the tissue block method is not suitable for primary culture of liver cells of the micropterus salmoides, and the digestion method can realize large-scale culture, and the digestion process can damage the primary liver cells and influence the growth activity of the liver cells to a certain extent. Disclosure of Invention The application aims to provide a culture medium suitable for culturing a liver cell line by a micropterus salmoides tissue block method. The invention provides a culture medium for culturing a liver cell line of micropterus salmoides, which comprises a primary culture medium; the primary culture medium is a DMEM/F-12 basic culture medium containing 18-22% fetal bovine serum, 4.5-5.5 mug/mL human FGF and 3.5-4.5% first antibiotic compound liquid; The first antibiotic compound liquid comprises 390-410U/mL penicillin, 0.38-0.42 mg/mL streptomycin and 180-220 mug/mL amphotericin. Preferably, the primary culture medium is a DMEM/F-12 basal culture medium containing 20% fetal bovine serum, 5 mug/mL human FGF and 4% first antibiotic compound solution; the first antibiotic compound liquid comprises 400U/mL penicillin, 0.4 mg/mL streptomycin and 200 mug/mL amphotericin. Preferably, the culture medium further comprises a subculture medium; The subculture medium is a DMEM/F-12 basal medium containing 18-22% fetal bovine serum and 1.8-2.2% second antibiotic compound liquid in volume concentration; The second antibiotic compound liquid comprises 180-220U/mL penicillin, 0.18-0.22 mg/mL streptomycin and 90-110 mug/mL amphotericin. Preferably, the subculture medium is a DMEM/F-12 basal medium containing the following concentration components of fetal bovine serum with the volume concentration of 20% and 2% of second antibiotic compound liquid; the second antibiotic compound liquid comprises 200U/mL penicillin, 0.2 mg/mL streptomycin and 100 mug/mL amphotericin. Preferably, the cell freezing solution is also included; the cell cryopreservation liquid is the cell cryopreservation liquid in the culture medium according to the technical scheme. The invention provides a method for establishing a liver cell line of micropterus salmoides, which comprises the following steps: primary culturing the liver tissue fragments of the micropterus salmoides in a primary culture medium of the culture medium to obtain a cell monolayer; And (3) after preparing single cells from the cell monolayer, placing the single cells in a subculture medium for subculturing to obtain the micropterus salmoides liver cell line. Preferably, the primary culture is carried out at a temperature of 27-29 ℃ for 10-15 days, wherein the primary culture is 5% CO 2; The primary culture is replaced once a day with primary culture medium. Preferably, the subculture is performed once every 2 days; The passage number of the subculture comprises 10-120 passages; and carrying out passage according to the ratio of 1:1 for 1 to 10 generations, and carrying out passage according to the ratio of 1:2 after 10 generations. Preferably, the micropterus salmoides liver cell line further comprises preservation in a cell preservation solution; the cell preservation solution comprises fetal bovine serum and DMSO in a volume ratio of 8-10:0.8-1.2. Preferably, the largemouth black bass liver cell line comprises cell resuscitation; The cell resuscitating comprises melting in a water bath, separating cells, and re-suspending in a subculture medium; The melting temperature of th