In-vitro culturing method for chlamys farrei hear cells

A technology for scallops and heart cells, which is applied to the field of in vitro culture of scallop heart cells, can solve the problems of difficulty in meeting the nutritional metabolism requirements of in vitro cultured cells, contamination of microorganisms and protozoa, and lack of mollusk culture medium, etc. Microbial contamination such as bacteria, no effect on cell viability, and low cost

Inactive Publication Date: 2010-12-01
OCEAN UNIV OF CHINA
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Problems solved by technology

[0003] Because most of the organs of marine bivalves are directly in contact with seawater, they are polluted by various microorganisms and protozoa, which often leads to incomplete tissue disinfection and contamination of primary culture; at the same time, there is no medium specially designed for molluscs, so it is difficult to meet the requirements of other species. The nutritional and metabolic requirements of cultured cells in vitro necessitate the establishment of a method for marine bivalves cell culture

Method used

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Embodiment 1

[0016] The heart tissue samples of Chlamys farreri were washed 3 times with boiled filtered seawater to remove microorganisms on the surface; special disinfectant for heart tissue of Chlamys farreri contained 500 units / ml penicillin, 500 micrograms / ml streptomycin, 100 units / ml Qing Amamicin and 2 μg / ml nystatin in boiled filtered seawater in water.

[0017] The preparation of Chlamys farreri heart cell culture medium of the present invention: first in the aqueous solution that has dissolved L15 medium powder, add NaCl 20.2 grams / liter, KCl 0.54 grams / liter, CaCl 2 0.60 g / L, MgSO 4 1 g / L and MgCl 2 3.9 g / L, filter and sterilize with a 0.22-micron microporous membrane, then add 5% fetal bovine serum, 6 mmol calcium ions, 2 mmol L-glutamine, 50 mmol taurine, 100 units / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml kanamycin, and 1 μg / ml nystatin; finally adjust the pH to 7.2. Soak the heart tissue in the above-mentioned special disinfectant for the heart tissue of Chlamys f...

Embodiment 2

[0019] The heart tissue samples of Chlamys farreri were washed 3 times with boiled filtered seawater to remove microorganisms on the surface; special disinfectant for heart tissue of Chlamys farreri contained 500 units / ml penicillin, 500 micrograms / ml streptomycin, 100 units / ml Qing Boiled and filtered seawater solution of damycin and 2 μg / ml nystatin; preparation of ctenophore heart cell culture medium: first add NaCl 20.2 g / L and KCl 0.54 g / L to the solution in which L15 medium powder is dissolved Liter, CaCl 2 0.60 g / L, MgSO 4 1 g / L and MgCl 2 3.9 g / L, filter and sterilize with a 0.22-micron microporous membrane, then add 5% fetal bovine serum, 6 mmol calcium ions, 2 mmol L-glutamine, 50 mmol taurine, 100 units / ml penicillin, 100 μg / ml streptomycin, 50 μg / ml kanamycin, and 1 μg / ml nystatin; finally adjust the pH to 7.4. Soak the heart tissue in the above-mentioned special disinfectant for the heart tissue of Chlamys farreri for 20 minutes, wash it with boiled filtered s...

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Abstract

The invention relates to an in-vitro culturing method for chlamys farrei hear cells. The method is characterized by cleaning a chlamys farrei hear tissue, soaking the chlamys farrei hear tissue in a disinfectant for 20 minutes, cleaning the chlamys farrei hear tissue with boiled filtered seawater, removing the chlamys farrei hear tissue into a shear liquid, shearing the chlamys farrei hear tissue into pieces with a volume of 1 cubic millimeter, uniformly distributing the pieces in a culture flask which contains 1.5ml of culture medium, culturing the chlamys farrei hear tissue in a biochemical incubator of which the temperature is 20 to 23 DEG C for 16 to 24 hours, replacing 2ml of culture medium each day until the chlamys farrei hear cells are emigrated, namely in-vitro cultured chlamys farrei hear cells are obtained, and replacing the culture medium every 3 to 7 days so as to keep the in-vitro cultured chlamys farrei hear cells normally grow. In the method, the cost is low and the efficiency is high; because a proper antibiotic is adopted in a primary cell culture process of the chlamys farrei hear cells, contamination of microorganisms, such as bacteria and the like, can be effectively overcome; and because multiple trophic factors maintaining cellular metabolism needs are added in the culture medium of the chlamys farrei hear cells, relatively long in-vitro cultivation of the chlamys farrei hear cells is realized.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a method for culturing heart cells of Chlamys farreri in vitro. Background technique [0002] Marine bivalves have high economic value and are an important part of my country's mariculture industry. However, in recent years, large-scale deaths of cultured shellfish in coastal areas of my country have occurred frequently, causing heavy economic losses to the shellfish industry. Most of the current research focuses on the level of electron microscopy, which can only be analyzed from the changes of cell structure, and it is difficult to obtain the exact pathogenic mechanism. The establishment of an in vitro culture system for marine bivalve cells will reveal the mechanism of host-pathogen interaction at the cellular and molecular levels, and build a suitable diagnostic tool. In addition, marine bivalve cell culture has great application prospects in the growth and reproductio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
Inventor 张志峰王华晏萌林娜邵明瑜
Owner OCEAN UNIV OF CHINA
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