Swordtail fish larva fish cell line and construction method and application thereof

A technology of swordtail larvae and a construction method, which is applied in the field of medical biology, can solve problems such as no reports on the swordtail larvae cell line, and achieve the effect of stabilizing the characteristics of the cell line

Active Publication Date: 2019-12-03
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The establishment of fish primary cell lines is currently a relatively mature technology, but there are still great chances and difficulties in obtaining cell lines that are sensitive to viruses and have gene expression that responds to pollutants. Strains need fish cell lines An effective way is to compare quality with quantity, that is, to prepare large quantities of primary cell lines, so as to screen out sensitive cell lines
[0010] So far, there have been reports on several cell lines such as the swordtail brain cell line and the swordtail embryo cell line, but there has been no report on the swordtail larvae cell line

Method used

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  • Swordtail fish larva fish cell line and construction method and application thereof
  • Swordtail fish larva fish cell line and construction method and application thereof
  • Swordtail fish larva fish cell line and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of SFF Cell Line of Swordtail Larvae

[0034] 1. Prepare cell culture medium:

[0035] Take the DMEM / F-12 medium of GIBCO Company, add fetal bovine serum accounting for 10-20% of the total volume used, 10%-20% swordtail brain cell growth medium, 20ng / mL basic fibroblast growth factor (bFGF ), 1ng / mL of epidermal growth factor (EGF), stored at 4°C, for later use;

[0036] 2 primary culture:

[0037] Take 5-6 freshly born swordtail larvae (viviparous), disinfect them in 75% ethanol for 5-10 seconds, rinse them with PBS and place them in a 5mL sterile penicillin vial containing 200ul of PBS. Cut it into pieces with scissors; add 3 to 5 times the volume of digestive solution A and mix evenly; evenly spread the broken tissue pieces mixed with digestive enzymes into the culture bottle whose outer wall is pre-coated with 0.1% gelatin; turn the culture bottle upside down Put in saturated humidity of 27℃, 5% CO 2 In the incubator, after 6-8 hours of inversion an...

Embodiment 2

[0043] Determination of Optimum Conditions for the Cell Line of Swordtail Larvae

[0044] 1. Determination of the best culture medium for swordtail larvae cell lines

[0045] Select DMEM / F-12, M199, DMEM / F-12 and L-15 mixed medium (1:1), L-15 four kinds of cell culture medium, and add FBS with a final concentration of 10% to prepare cell culture medium. Adjust the cell density to 5 x 10 4 mL -1 , each of the four mediums was inoculated in a 6-well plate at an amount of 2.5 mL / well, and cultured in an incubator at 27°C. Cells in 3 wells were taken out from each experimental group every 1 day, collected and counted by Trypsin-EDTA digestion method, co-cultured for 7 days, counted 7 times continuously, and the growth curve was drawn. Determine its optimum medium is DMEM / F-12 and L-15 mixed medium (1:1) ( figure 2 A).

[0046] 2. Determination of optimal serum concentration of swordtail larvae cell line

[0047] Prepare culture solutions with FBS concentrations of 5%, 10%, ...

Embodiment 3

[0050] Cell Freezing and Recovery

[0051] 1. Cryopreservation of cells

[0052] Collect 1 bottle (25cm 2 ) in the logarithmic growth phase of the swordtail larvae cells, centrifuged at 1000g for 5min, discarded the supernatant, and mixed with 1mL cryopreservation solution (DMEM / F-12 containing 20% ​​FBS and 10% DMSO and L-15 (1 :1) Culture medium) resuspend the cells and place them in cryopreservation tubes, place the cryopreservation tubes in a programmed cooling box at -80°C overnight, and finally put them into liquid nitrogen (-196°C) for long-term storage, and prepare well Record.

[0053] 2. Recovery of cells

[0054] When resuscitating the cells, take out the cryopreservation tube from the liquid nitrogen, thaw it quickly in a water bath at 37°C, centrifuge at 1000g for 5 minutes to collect the cells, and inoculate them in a 25cm 2 Place in a culture flask in an incubator at 27°C for culture. After the cells adhere to the wall, discard the supernatant, replace the c...

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PUM

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Abstract

The invention discloses a swordtail fish larva fish cell line. The swordtail fish larva fish cell line is constructed by primary culture and subculturing. The invention further provides an applicationof the swordtail fish larva fish cell line to separation of aquatic product animal viruses and environmental pollutant monitoring. Swordtail fish larva fish is subjected to cell culture, the cell culture condition is primarily determined, chromosome analysis is performed, the sensibility of various aquatic animal viruses by cells is detected, and the inductive effect of antibiotic environmental pollutants namely rifampicin on cells is researched. The cell line establishes foundation for research of later stage aquatic animal viruses and application of the cell line as an in vitro toxicology cell model to toxicology correlational research of an environmental pollutant.

Description

technical field [0001] The invention relates to a cell line and its construction method and application, in particular to a swordtail larvae cell line, its construction method and application, and belongs to the field of medical biotechnology. Background technique [0002] Swordtail fish (Xiphophorus helleri, swordtail fish) is a tropical and subtropical small fish, and its taxonomic status is Osteichthyes, Cyprinodontiformes, Poeciliidae, and Xiphophorus. Swordtail fish are small in size, viviparous, short in reproductive cycle, convenient for feeding and management in the laboratory, and are sensitive to various pesticides, heavy metals and other poisons. They are ideal for water environment monitoring, toxicity testing and aquatic animal virus detection. Experiment with aquatic animals. [0003] As the cell model of fish tissue, fish cells are widely used in the field of biomedical research and the detection and toxicity research of environmental pollutants, laying the f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/6888
CPCC12N5/0602C12N2509/00C12Q1/6888C12Q2600/158
Inventor 王英英王庆曾伟伟李凯彬李莹莹尹纪元石存斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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