Building method of fish head and kidney tissue derived cell lines

A technique for establishing a method and kidney tissue, which is applied in the field of fish cell lines, and can solve problems such as the absence of reports on cell lines derived from head kidney

Inactive Publication Date: 2013-04-24
沙珍霞
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] So far, although there are literatures reporting semi-smooth tongue sole embryonic cell line (Yellow Sea Fisheries Research Institute of China Fisheries Research Institute, Sha Zhenxia, ​​Acta Oceanologica Sinica.vol.29.No.2:81-87), cardiomyocyte cell line ( Yellow Sea Fisheries Research Institute, Wang Xianli, Fish Physiol Biochem 36:1181–1189), liver cell line (Yellow Sea Fisheries Research Institute of China Fisheries Research Institute, Ren Guocheng, High technology communication.18 (6): 657-660), testicular cell line (Yellow Sea Fisheries Research Institute of China Fisheries Research Institute, Zhang Bo, Int.J.Biol.7(4):452-459) and other 4 cell lines were established, but there is no report of head kidney-derived cell lines

Method used

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  • Building method of fish head and kidney tissue derived cell lines
  • Building method of fish head and kidney tissue derived cell lines
  • Building method of fish head and kidney tissue derived cell lines

Examples

Experimental program
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Embodiment 1

[0027] Example 1. Method for establishing cell line derived from head kidney of tongue sole

[0028] 1. Prepare cell culture medium: take 9.6 grams of GIBCO MEM medium and 4.76 grams of Hepes, fully dissolve and mix for 4 hours, adjust the pH to 7.0-7.3 with NaOH, then filter with suction, aliquot, and store at 4°C; use Add 10-20% of the total volume of fetal bovine serum, 1mM sodium pyruvate, 50mM 2-mercaptoethanol, 10ng / ml human basic fibroblast growth factor, 100 U / ml penicillin, 100 U / ml Streptomycin, stored at 4°C.

[0029] 2. Primary culture: Take the head kidney tissue of tongue sole weighing about 250 grams, place it in an autoclaved glass plate, wash it twice with PBS, and cut the kidney tissue into about 1 mm 3 Add 1ml of 0.25% trypsin and digest at room temperature for 20 minutes. Add 3 ml of cell culture medium to stop the digestion, collect the digested tissue cell suspension into a 15 ml centrifuge tube, centrifuge at 2200 rpm for 5 minutes, and remove the su...

Embodiment 2

[0031] Example 2. Identification method of cell line derived from head kidney tissue of half-smooth tongue sole

[0032] 1. Cell cryopreservation and recovery

[0033] 1) Cell cryopreservation: Select a bottle of cells that are vigorously growing, in the exponential growth phase, and have a cell density above 90%, add 1 ml of 0.25% trypsin to digest for 2 minutes, absorb the digestive juice, and use 2 ml of cryopreservation solution ( Cell culture medium containing 10% dimethyl sulfoxide) to suspend cells, transfer to cryopreservation tubes, store at 4°C for 30 minutes, store at -80°C for 12 hours, then transfer to liquid nitrogen for storage, and make records.

[0034] 2) Recovery of cells: Take out the cryopreservation tube from liquid nitrogen, put it in a 40°C water bath to thaw quickly, centrifuge at 2200 rpm for 5 minutes, discard the supernatant, add 2 ml of cell culture medium to suspend the cells, and transfer to a culture bottle Place in an incubator at 24°C for c...

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Abstract

The invention discloses a building method of fish head and kidney tissue derived cell lines and the obtained cell lines. The cell lines are obtained by performing a primary culture and a subculture for the treated fish heads and kidney tissues, wherein an employed cell culture medium is prepared by adding fetal calf serum, sodium pyruvate, 2-mercaptoethanol and a human basic fibroblast growth factor into a basic cell culture medium.

Description

technical field [0001] The invention relates to a method for culturing fish cell lines in vitro, and belongs to the technical field of biological cell culture. Background technique [0002] In vitro cultured fish cell lines are important tools widely used in virology, immunology, toxicology, developmental biology, oncology genomics, genetics, and resource conservation. Up to now, more than 280 different fish cell lines have been established successively, playing an important role in the fields of physiology, virology, toxicology, tumor and genetic engineering. [0003] Half-smooth tongue sole ( Cynoglossus semilaevis Gunther) is mainly distributed in the Bohai Sea and the Yellow Sea in my country. Its meat is delicate, delicious, rich in nutrition, high in economic value, and less in natural resources. Since the success of artificial breeding in 2003, half smooth tongue sole has become the main cultured species in northern my country in recent years, with an annual output...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12R1/91
Inventor 沙珍霞陈松林郑媛王娜谢明树
Owner 沙珍霞
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