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Culture medium and method for culturing chlamys farreri trochophore cell lines

A technology for Ctholecystis scallops and dandelion larvae is applied in the field of culture medium for cultivating scallop dandelion larvae cell lines. improve survival

Inactive Publication Date: 2016-05-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a culture medium and method for cultivating the trochophore cell line of Chlamys farreri, aiming to solve the problem that the subculture of shellfish cells cannot be realized in the prior art

Method used

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  • Culture medium and method for culturing chlamys farreri trochophore cell lines
  • Culture medium and method for culturing chlamys farreri trochophore cell lines
  • Culture medium and method for culturing chlamys farreri trochophore cell lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of Chlamys farreri troponin larvae cell line

[0027] 1) Configure culture medium

[0028] Preparation of scallop serum: extract hemolymph of scallop scallop from the adductor muscle, place it in a sterile container at 4°C overnight, centrifuge the supernatant at 2000g for 10min the next day, collect the supernatant and pass through 0.45μm And 0.22μm pore size millipore filter filter sterilization. Store at -20°C after aliquoting.

[0029] Preparation of yolk extract from Chlamys farreri: Mature Chlamys scallop ovaries were dissected, washed 3 times in sterilized seawater, and shredded with scissors until they were chyle-shaped. Take 1ml of chylous tissue into a 15ml centrifuge tube, add 10ml of calcium and magnesium ion-free phosphate buffer; ultrasonically break on ice for 3min (super 15s and stop for 10s), power 200W. After the end, let it stand for 5 minutes, then centrifuge at 8000g at 4°C for 1 hour; collect the supernatant and sterilize ...

Embodiment 2

[0039] Example 2 Characteristic Analysis of Chlamys farreri Troponilar Cell Lines

[0040] 1) Morphological description

[0041] Microscopic photos of the 10th and 50th passages of trochophore cells of Chlamys farreri image 3 and Figure 4 As shown, the shape of the cells is nearly round, and the Giemsa staining microscope photos of the 10th passage of the trochophore cells of Chlamys farreri are as follows: Figure 5 As shown, it shows that the nucleoplasmic ratio is large, the cell proliferation is vigorous, and there are clonal clusters locally.

[0042] 2) Growth curve

[0043] Take the 45th passage trochophore logarithmic phase cells with stable proliferation and passage once every 6 days, and adjust the cell density to 5×10 4 / ml, inoculated in a 24-well plate at 1ml / well for culture. Take out 3 wells every 24h and count, carry out continuously 10d, take culture time (d) as abscissa, take cell concentration as ordinate (cell / ml), carry out the drawing of growth cur...

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Abstract

The invention discloses a culture medium and method for culturing chlamys farreri trochophore cell lines. The culture medium comprises the following components: 13.7 g / l of an L-15 base culture medium, 20.2 g / l of NaCl, 0.54 g / l of KCl, 0.60 g / l of CaCl2, 1 g / l of MgSO4, 3.9 g / l of MgCl2 and fetal bovine serum with the final concentration of 5%, 2 mmol / l of glutamine, 1% of a chlamys farreri yolk extract liquid, 1% of chlamys farreri serum, 100 IU / ml of penicillin, and 100 [mu]g / ml of streptomycin, wherein the pH is 7.2-7.4. The culture method comprises that the culture medium is used for performing single cell primary culture of trochophores, to realize early passage and serial subcultivation. The culture medium can effectively increase cell survival, and makes cells continuously proliferate; and the method has guiding significance on establishment of other marine shellfish cell lines, and has great application value.

Description

technical field [0001] The invention belongs to the technical field of cell culture of marine shellfish, and in particular relates to a culture medium and a method for cultivating the trophoid larva cell line of Chlamys farreri. Background technique [0002] Cell culture is an important means and one of the commonly used methods in bioengineering. It has been widely used in research fields such as chromosome analysis, cell metabolism, gene expression, immunity, carcinogenesis mechanism, virus culture, isolation, identification and drug screening. Shellfish tissue culture work began in the late 1960s, and more than a dozen kinds of oysters, mussels, clams, etc. have been reported. Domestic research in this field began in the 1980s. The key to the success of cell culture is whether or not a suitable environment can be created so that cells in vitro can proliferate continuously. At present, there is no cell culture medium for marine shellfish. In order to make shellfish cells ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12R1/91
Inventor 张志峰季爱昌李学玉秦贞奎马晓世杨丹丹
Owner OCEAN UNIV OF CHINA
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