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In-vitro culture method of adipocytes of large yellow croaker

An adipocyte and in vitro culture technology, applied in the field of cell culture, can solve the problems such as the inability to directly use large yellow croaker in culture conditions and temperature, the inability to directly use large yellow croaker in physiological state and culture parameters, and the difficulty.

Inactive Publication Date: 2013-05-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In 2003, Vegusdal et al. successfully cultured Atlantic salmon adipocytes in vitro for the first time, and then in 2006, Oku et al. Pagrus major ) fat cell culture, in 2007, Liu Qian did the fat cell culture in grass carp, but at present, as my country's important economic marine fish - the fat culture of large yellow croaker has not been reported yet
Atlantic salmon is a cold-water animal, and its culture conditions and temperature cannot be directly applied to large yellow croaker. Red sea bream uses serum-free culture technology, which is currently quite difficult to apply to large yellow croaker. Grass carp is a freshwater herbivorous fish, and its physiological state and culture parameters can not be directly used in large yellow croaker

Method used

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  • In-vitro culture method of adipocytes of large yellow croaker
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  • In-vitro culture method of adipocytes of large yellow croaker

Examples

Experimental program
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Embodiment 1

[0045] Embodiment 1, a kind of large yellow croaker adipocyte culture method in vitro, carries out following steps successively:

[0046] 1) Take the adipose tissue from the abdominal wall of the large yellow croaker:

[0047] Take a large yellow croaker weighing 300-500g, first anesthetize it with MS-222, cut off the gill arch, bled the fish, and hit the head until the fish is dead; under aseptic conditions, cut open the abdominal cavity of the large yellow croaker, and take the fat tissue of the abdominal wall.

[0048] 2) The above-mentioned adipose tissue is subjected to cell dispersion treatment, and the following steps are performed in sequence:

[0049] A. Cut the adipose tissue into 1mm 3 For the left and right small pieces, soak and wash with PBS buffer containing double antibodies (100IU / ml penicillin, 100IU / ml streptomycin); get small pieces of tissue after washing;

[0050] B. Suspend the rinsed small pieces of tissue in 0.2% collagenase II digestion solution, an...

Embodiment 2

[0056] Example 2. A method for in vitro culture of large yellow croaker adipocytes. When the primary cultured cells described in step 4) of Example 1 reach 70-80% confluence, subculture inoculation and culture are carried out. The specific content is to carry out the following steps in sequence :

[0057] ①. When the primary cultured cells reach 70-80% confluence, first digest with 1ml trypsin digestion solution at 25°C for 2-3 minutes. The preparation method of the trypsin digestion solution is as follows: mix 0.25g trypsin with 100ml PBS buffer was mixed to prepare 0.25% trypsin digestion solution. 1 bottle (for the bottom area is 25cm 2 Petri dish) the cells were digested with 1ml of trypsin solution.

[0058] Then stop the digestion with the same volume of complete medium as the trypsin digestion solution, and centrifuge at 1000rmp / min for 5min to obtain a precipitate; subculture the precipitate and complete medium at a volume ratio of 1:3; during this culture process, e...

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Abstract

The invention discloses an in-vitro culture method of the adipocytes of large yellow croaker. The method comprises the following steps: 1) taking fat tissue from the abdominal wall of a large yellow croaker; 2) performing cell dispersing treatment to the fat tissue to obtain the preadipocytes of the large yellow croaker; 3) inoculating the preadipocytes in complete medium, blowing and beating evenly to prepare cell suspension, wherein the complete medium is the culture solution containing 20% of fetal calf serum; and 4) performing primary culture: culturing the cell suspension at 24-26 DEG C to obtain preadipocytes growing on a surface. When 70-80% of the preadipocytes obtained in the step 4) converge, subculturing inoculation can be performed. By adopting the method disclosed by the invention, the in-vitro culture of the fat cells of large yellow croaker can be effectively realized.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a method for culturing large yellow croaker adipocytes in vitro. Background technique [0002] my country is the largest aquaculture country in the world, and the excessive accumulation of fat in farmed fish is one of the main problems that plague the aquaculture industry. Large yellow croaker[ Pseudosciaena crocea (Richardson)] is an important economic fish in my country. Due to overfishing, the production has declined sharply. In 1987, the artificial breeding of large yellow croaker in my country was successful, and artificial breeding began in 1994. However, the size of farmed large yellow croaker is poor, and the taste and nutritional value of the fish meat are far inferior to those of wild large yellow croaker (Duan Qingyuan et al., 2000). Therefore, it cannot meet the market's "quality" demand. A large number of studies have shown that excessive fat deposition is an important fact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
Inventor 王新霞汪以真
Owner ZHEJIANG UNIV