Separation culture method of primary hepatocyte of jian carp

A technology for the isolation and cultivation of primary hepatocytes, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of small number of hepatocytes, long separation time, and many red blood cells, so as to save separation time and meet Experimental requirements, simple and fast results

Inactive Publication Date: 2015-01-21
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, regarding the isolation and culture methods of fish hepatocytes, the number of hepatocytes obtained is small, the separation time is long, the red blood cells are too many, and the activity is poor, which cannot be well applied in experimental research.

Method used

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  • Separation culture method of primary hepatocyte of jian carp
  • Separation culture method of primary hepatocyte of jian carp
  • Separation culture method of primary hepatocyte of jian carp

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Experimental program
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Embodiment 1

[0032] Disinfection of experimental materials and instruments:

[0033] The L-15 medium, rat tail collagen, fetal bovine serum, double antibody, 0.25% trypsin digestion solution, 75% alcohol, 0.4% trypan blue and other reagents and medicines required for the experiment, scissors, tweezers, pipette tip, Sterilize pipettes, Erlenmeyer flasks, glass beads, filters, counting plates, etc. (The percentages are volume percentages)

[0034] Cell culture plate pretreatment:

[0035] The purpose of cell culture plate pretreatment is to promote cell adherence, take out the cell culture plate, drop 200 μL of rat tail collagen solution into each well under sterile conditions, and gently shake it to evenly cover the bottom of the well. Put the culture plate coated with collagen solution into CO 2 Incubator (27°C, 5% CO 2 ) overnight. The next day, take out the culture plate to the ultra-clean workbench, discard the collagen solution in each well, and then add 500 μL L-15 medium to wash...

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Abstract

The invention discloses a separation culture method of the primary hepatocyte of a jian carp. The separation culture method comprises the following steps: selecting a healthy jian carp without injury, collecting blood from the caudal vein of the jian carp, then sterilizing the surface of a fish body, placing into a super clean bench, and aseptically collecting the liver of the jian carp; rinsing by using a PBS solution which contains double antibodies, adding a trypsin digestion solution, wherein the temperature of digestion treatment is 26-28 DEG C, and the time of the digestion treatment is 15-20 minutes; after digestion is finished, adding to a culture medium A to finish the digestion, filtering the trypsin digestion solution by using a filter screen of 200 meshes, and collecting filter liquor; and respectively centrifugalizing 50 grams of the filter liquor and 30 grams of the filter liquor for 5 minutes, then washing twice by using a culture medium B, removing a supernatant to obtain a precipitation, namely an extracted hepatocyte, preparing a cell suspension, and planking for culture. The experimental method disclosed by the invention is simple and fast and can save the separation time to a great extent. The separation time adopted by an experiment is about 40 minutes, and the obtained hepatocyte has the advantages of good growth condition, small erythrocyte quantity and cell survival rate of about 95%.

Description

technical field [0001] The invention relates to a method for separating and culturing primary hepatocytes of Jian carp, belonging to the field of cell biology and biotechnology. Background technique [0002] The culture of primary hepatocytes originated in the 1940s. The successful culture of hepatocytes has been applied in many fields, such as hepatocyte transplantation and bioartificial liver. Regarding the separation method of hepatocytes, mammals have made rapid progress. Two-step collagenase perfusion method, tissue block culture method, enzyme digestion method, etc.; while the isolation and culture of fish hepatocytes started late and progressed slowly, two-step perfusion method and enzyme digestion method were mainly used. Because hepatocytes have a complete drug-metabolizing enzyme system and are the main site for drug biotransformation, the in vitro culture of hepatocytes is playing an increasingly important role in the study of liver diseases. Primary hepatocytes c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 杜金梁曹丽萍殷国俊贾睿刘英娟申玉金赵才源
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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