Method for culturing and inducing tilapia mossambica peritoneal preadipocytes and culture medium thereof

A technology for inducing culture medium and preadipocytes, which is applied in the cultivation process, animal cells, tissue culture, etc., to achieve the effect of broad application prospects, convenient promotion and application, and realization of in vitro subculture

Inactive Publication Date: 2017-02-22
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the establishment of tilapia adipocyte culture system has not been reported

Method used

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  • Method for culturing and inducing tilapia mossambica peritoneal preadipocytes and culture medium thereof
  • Method for culturing and inducing tilapia mossambica peritoneal preadipocytes and culture medium thereof
  • Method for culturing and inducing tilapia mossambica peritoneal preadipocytes and culture medium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] The present embodiment is exemplified by "in vitro extraction and cultivation of tilapia (Tilapia) abdominal adipocytes"

[0080] 1. Experimental animals:

[0081] Tilapia (700-900g) that has just died (it can also be purchased from the market, is healthy and has no body surface damage)

[0082] 2. Experimental steps:

[0083] (1) Bleeding by cutting off the gill arch (if the tilapia is healthy, the tilapia should be anesthetized with MS-222);

[0084] (2) Under sterile conditions, quickly separate the adipose tissue around the mesentery, put it directly into the Krebs-Henseleit buffer, wash it three times, cut it into pieces and place it in a centrifuge tube, then add an equal volume of 1% type II collagenase to the 28 ℃ water bath shaking digestion for 1h;

[0085] (3) filter the suspended fat cells with 100 mesh nylon membrane, remove connective tissue, and wash three times;

[0086] (4) Add erythrocyte lysate to remove blood cells, resuspend cells in DMEM / F12 me...

Embodiment 2

[0100]Experimental steps (1)~(3) are with embodiment 1;

[0101] In step (4), red blood cell lysate was added to remove blood cells, cells were resuspended in DMEM / F12 medium containing fetal bovine serum, and seeded in cell plates at 28°C, 5% CO 2 Cultivate under conditions, change the medium every 4 days, and cultivate for 14 to 20 days;

[0102] (5) The cultured preadipocytes were digested with 0.12% trypsin, resuspended, and mixed with 2~3×10 5 cells / ml inoculated in the proliferation medium, cultured for 2-3 days, the cells can cover the cell plate.

[0103] In this embodiment, the cells that have been successfully passaged can continue to proliferate, and can cover the entire bottom of the cell plate in 2 to 3 days.

[0104] Figure 5 In order to pass the cells cultured in step (4) to a new cell plate, use 2~3×10 5 cells / ml inoculated, cultured for 2-3 days, the cells can cover the bottom of the entire cell plate.

Embodiment 3

[0106] Other conditions are the same as in Example 1. When the induction medium in steps (4) to (5) is the grass carp induction medium, the lipid droplets produced by the cells are not obvious, such as Figure 6 The figure shows that when the grass carp induction medium was induced for 12 days, the oil red staining showed that the lipid droplets in the cells were blurred, and almost no clear lipid droplets were produced in the cells. Neutral fat cells almost disappeared.

[0107] The composition of the grass carp induction medium is DMEM / F12 medium with 15% fetal bovine serum, 20 μg / ml insulin, 1 μmol / L dexamethasone, and 10 nmol / L triiodothyronine.

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Abstract

The invention discloses a method for culturing and inducing tilapia mossambica peritoneal preadipocytes. The method comprises the following steps: a) resuspending cells by using a DMEM/F12 culture medium with 10%-15% of fetal calf serum, inoculating the cells into a cell plate, and culturing for 7 days at the temperature of 20-30 DEG C under the condition of 5% CO2; and b) replacing an induction culture medium for sequentially culturing, after inducing for 7-14 days, carrying out oil red O staining of the cells, wherein the fuzzy cell membrane edges can be seen under the microscope, and the interiors of the cells are stained to be red lipid droplets. The invention also discloses a passage method of tilapia mossambica peritoneal preadipocytes. A proliferation culture medium is used for culturing the preadipocytes for 14-20 days, then the preadipocytes are inoculated according to 2 *10<5> to 3*10<5> per milliliter, and the cells can overgrow on the cell plate after the cells are cultured for 2-3 days. The method is capable of successfully carrying out in-vitro culture, induction and passage of the tilapia mossambica peritoneal preadipocytes; the technical reference is provided for the research of the tilapia mossambica lipid metabolism.

Description

technical field [0001] The invention relates to the technical field of aquatic animal nutrition, in particular to a method for cultivating and inducing tilapia preabdominal adipocytes. Background technique [0002] Since Wolf et al. established the first fish cell line, the rainbow trout (Oncorhynchusmykiss) gonad cell line RTG-2 in 1962, fish cell culture technology has been continuously developed and matured, from the initial use for the isolation of fish viruses, From the identification and preparation of virus vaccines to the current applications in environmental toxicology, fish oncology, physiology, endocrinology, genetics and resource protection, fish cell culture technology has been widely used. The main reason is that, as an emerging technology, fish cell culture has unique advantages that live fish culture cannot match: (1) low cost; cell line culture and preservation do not require many human resources, and cell culture has a high degree of standardization and no ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0653C12N2500/40C12N2501/33C12N2501/39C12N2501/998C12N2501/999
Inventor 杜震宇王雅文乔芳
Owner EAST CHINA NORMAL UNIV
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