Construction method of bastard halibut brain cell system

A technology of cell line and establishment method, applied in the field of marine fish cell culture, can solve the problems such as the establishment of a cell line with no brain tissue, and achieve the effects of easy mastery and operation, easy operation and strong repeatability

Inactive Publication Date: 2014-05-14
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there are only flounder fin ray cell lines at home and abroad (Ocean University of China, Tong Shangliang, Aquaculture, 1997, 156:327-333.), embryonic cell lines (Yellow Sea Fisheries Research Institute of China Fisheries Research Institute, Chen Songlin, Diseases of Aquatic Organisms, 2004, 60: 241-246.), kidney cell line (Huanghai Fisheries Research Institute, China Fisheries Research Institute, Wang Na, Journal of Fish Diseases, 2011, 34: 81-85.), etc. Cell lines, there is no establishment of brain tissue cell lines of this fish species

Method used

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  • Construction method of bastard halibut brain cell system
  • Construction method of bastard halibut brain cell system
  • Construction method of bastard halibut brain cell system

Examples

Experimental program
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Effect test

Embodiment 1

[0026] A method for establishing a cell line derived from flounder brain, the steps are as follows:

[0027] 1) Preparation of cell culture medium: take Hyclone's MEM culture medium, add 20% of the total volume of fetal bovine serum to the culture medium, 100U / mL penicillin, 100μg / mL streptomycin, 10ng / mL human basic fibroblasts Growth factors, pH 7.0-7.4, stored at 4°C, for later use.

[0028] 2) Primary culture: Aseptically take brain tissue of 250g flounder, and place it in a sterile glass plate with 4mL Hyclone MEM culture solution added with 400U / mL penicillin and 400μg / mL streptomycin for 3-5min. Aspirate the culture medium, wash the brain tissue twice with 1-1.2mL PBS (pH7.2), aspirate the PBS, and cut the brain tissue into about 1mm 3 For small pieces of brain tissue, add 1mL of the above step 1) to prepare cell culture medium to suspend the small pieces of brain tissue, and then transfer to 25cm 2 In a culture bottle, culture it upright in an incubator at 25±0.2°C. ...

Embodiment 2

[0031] Identification and application of flounder brain cell line

[0032] 1) Cryopreservation and recovery of cells

[0033] Freezing of cells:

[0034]Select the subcultured brain cells that are in the exponential growth phase and have a cell density above 90%, and digest them according to the conventional method: suck out the culture medium, rinse with PBS (pH7.2), suck out the PBS, and add 1 mL of 0.25% trypsin solution for digestion. When the cell shrinkage is observed under the microscope, the trypsin solution is discarded, and the observation is continued under the microscope. When the cells become round, add 2 mL of fresh above-mentioned embodiment step 1) to the original bottle to prepare the cell culture medium to prepare the cell suspension. Transfer the cell suspension to a 15mL centrifuge tube at 2200r p m Centrifuge for 2min, remove the supernatant. Suspend the cells with 2 mL of pre-cooled cryopreservation solution (i.e. MEM cell culture medium from Hyclone C...

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Abstract

The invention discloses a method for establishing a bastard halibut brain tissue-derived cell system. The method comprises brain tissue primary culture, subculture, cryopreservation, recovery and identification, wherein cell culture fluid adopted is obtained by adding fetal calf serum and human basic fibroblast growth factor to basal cell culture fluid. The established bastard halibut brain cell system is represented as an epithelioid-like astrocytic cell, which can support continuous passage to provide a great amount of bastard halibut brain cells and can be directly used for research of bastard halibut functional gene. The construction method disclosed by the invention not only is expected to serve as a platform for theoretical research of bastard halibut molecule cellular level but also can be used as an ideal material for research on fish hemadenology, reproductive biology and environmental internal-secretion interfering-substance toxicology.

Description

technical field [0001] The invention relates to seawater fish cell culture technology, in particular to a method for constructing a flounder brain cell line. Background technique [0002] The construction and cultivation of animal cell lines has long been a powerful tool for the study of developmental biology, endocrinology, toxicology, genetics, virology, and immunology. Up to now, nearly 280 different fish cell lines have been established successively, playing an important role in many fields such as physiology, virology, toxicology, tumor and genetic engineering. But relatively speaking, there are few cell lines of seawater fish, including only about 100 strains of saltwater fish. [0003] Fish brain tissue is an important tissue and organ of fish, and all its life activities are regulated and controlled by the nervous system and endocrine system, such as gonad differentiation, development, maturation, gamete discharge and other reproductive activities are regulated by t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12R1/91
Inventor 尤锋郑媛彭丽敏邹玉霞吴志昊谭训刚焦爽
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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