In vitro culture method for scatophagus argus kidney cells

A culture method and kidney cell technology, applied to artificial cell constructs, animal cells, vertebrate cells, etc., to achieve strong repeatability and good results

Inactive Publication Date: 2012-08-15
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • In vitro culture method for scatophagus argus kidney cells
  • In vitro culture method for scatophagus argus kidney cells
  • In vitro culture method for scatophagus argus kidney cells

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 Preparation medium

[0020] 1. Basal medium

[0021] Dissolve one packet of L-15 dry powder medium in 800ml of ultrapure water, add 5ml of HEPES (1M) buffer, stir well, adjust the volume to 1L, adjust the pH to 7.4, sterilize with a 0.22μm filter membrane, 4°C Save for later.

[0022] 2. Complete Medium

[0023] Primary medium: Take 100ml of the above basal medium, add 20% FBS, 400U ml -1 Penicillin and 400ug ml -1 streptomycin.

[0024] Primary medium containing bFGF: take 50ml of the above primary medium, add 10ng ml -1 bFGF, formulated as the primary culture medium containing bFGF.

[0025] Subculture medium: use the basal medium prepared above, add 15% and 10% FBS, 100U ml -1 Penicillin and 100ug ml -1 Streptomycin, used up within two weeks.

Embodiment 2

[0026] Example 2 Primary culture of money fish kidney cells

[0027] 1. Sterile kidney extraction

[0028] Take 1-year-old healthy money fish, cut the tail and bleed. The specific operation is as follows: soak the fish body in 75% alcohol for 30s, dissect and remove the kidney tissue in an ultra-clean workbench, and add 400U ml -1 Penicillin and 400ug ml -1 Streptomycin basal medium Wash kidney tissue to remove surface bacteria and blood, in order to ensure tissue sterility, soak kidney tissue in L-15 basal medium containing 5% chlorhexidine, 4% penicillin and streptomycin mixed solution For 5 minutes, cut the tissue to 1mm with ophthalmic scissors 2 size.

[0029] 2. Digestion of isolated kidney cells

[0030] Transfer the shredded kidney tissue to a 15ml centrifuge tube, add 5-6 ml of 0.25% trypsin, digest in a water bath at 28°C for 20 minutes, shake constantly to speed up the separation, and then add complete medium containing 20% ​​FBS Terminate the trypsinization...

Embodiment 3

[0033] Example 3 Subculture of money fish kidney cells

[0034] The primary kidney cells covered 80% of the bottom of the bottle and began to be passaged. The specific method of subculture is as follows: first suck out the medium, add 1ml 0.25% trypsin to wash the bottom of the bottle, then pour it out, then add 0.5ml 0.25%-EDTA, place it in a 28°C incubator for 1-2min, and observe the cell changes under the microscope. When round, immediately add 5ml of fresh medium, gently blow the bottom of the bottle to make the adherent cells fall off, the ratio of 1:1 for the first passage, and the ratio of 1:2 for passage thereafter. The concentration of FBS in the 1-10th generation cell culture medium was 15%, and decreased to 10% after the 11th generation.

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Abstract

The invention relates to an in vitro culture method for scatophagus argus kidney cells. The culture method comprises the following steps of: (1) preparing an L-15 basic culture medium, a primary culture medium, a basic fibroblast growth factor (bFGF)-containing primary culture medium and a subculture medium; (2) performing primary culture on the scatophagus argus kidney cells: a, acquiring a sterile kidney, b, digesting and separating the kidney cells, and c, inoculating; and (3) performing subculture on the scatophagus argus kidney cells. According to the invention, the adopted scatophagus argus kidney cell culture method is strong in repeatability and good in effect; and by reference and improvement on the conventional saltwater fish cell culture method, the culture method suitable for scatophagus argus kidney cell culture is groped, a stable kidney cell culture system is established, a scatophagus argus kidney cell line is established for the first time, a cell model is expected to provide for osmotic pressure regulation of scatophagus argus, and deep research of functional genomes of the scatophagus argus is facilitated.

Description

technical field [0001] The invention relates to a method for culturing fish cells in vitro, in particular to a method for culturing kidney cells in vitro. Background technique [0002] The salinity of seawater and freshwater is very different, but the salinity of the body fluids of fish inhabiting these two different waters is almost the same. pressure regulation. The ability of fish to regulate osmosis determines their adaptation to the salinity of the water environment. Although osmoregulation is very important for the survival of fish, so far, there is still a lack of in-depth research on its mechanism. At present, the main research on osmotic pressure regulation is focused on observing changes in gill filaments and chloride cells, Na / K-ATPase activity and hormone changes. In fact, this process is controlled by a complex signal transduction network in organisms. With the development of molecular biology, the mechanism of osmotic pressure regulation will be studied at t...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 张俊彬张莹莹
Owner SHANGHAI OCEAN UNIV
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