Continuous culture method of prawn cells

A culture method and shrimp technology, applied in the field of tissue culture and animal cells, can solve problems such as poor results, and achieve the effects of avoiding hypotonic rupture, alleviating the process of blackening, and ensuring precipitation and recovery.

Active Publication Date: 2019-09-27
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current effect of 3D culture and subculture of shrimp cells is not good

Method used

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  • Continuous culture method of prawn cells
  • Continuous culture method of prawn cells
  • Continuous culture method of prawn cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Preparation of matrigel for 3D culture of prawn cells

[0023] The matrices used are Basement Membrane MatrixPhenol Red Free (Cat. NO. 356237).

[0024] Specific steps are as follows:

[0025] (1) Melting of Matrigel. Place the centrifuge tube containing Matrigel in a refrigerator at 4°C overnight in an ice bath, melt and shake well;

[0026] (2) Laying of Matrigel. Pre-cool the culture plate and tip on ice, at 55-80μl / cm 2 Add matrigel to the culture plate in the ratio of ;

[0027] (3) After the Matrigel was flattened naturally, the culture plate was placed in a 37°C incubator to solidify for 30 minutes, and then used.

[0028] The addition ratio of Matrigel mentioned above is the result of repeated optimization. After the added Matrigel is naturally flattened, it does not fill the bottom of the entire culture well, but there is still a certain gap between it and the side wall of the culture well. . However, a large enough Matrigel platform can be ...

Embodiment 2

[0029] Example 2 Isolation and 3D culture of shrimp peripheral blood lymphocytes.

[0030] The specific steps of isolation and 3D culture of shrimp peripheral blood lymphocytes are as follows:

[0031] (1) Treat the prawns in boiled and sterilized seawater containing 1000IU / mL penicillin and 1000μg / mL streptomycin for 12-24h;

[0032] (2) Before extracting the hemolymph, soak the prawns in 75% alcohol for disinfection for 3-5 minutes to anesthetize them;

[0033] (3) Use iodophor cotton balls and 75% alcohol cotton balls to wipe the sampling site successively, that is, the pleural sinuses under the abdomen of prawns;

[0034] (4) After absorbing about 0.2-0.5mL of the complete culture medium of the prawn cells with a 1-mL syringe, extract the hemolymphocytes of the prawns from the pleural sinuses under the abdomen of the prawns, and mix them immediately;

[0035] (5) After the blood is drawn, the needle of the syringe is removed, and the prawn blood lymphocyte suspension in ...

Embodiment 3

[0045] Embodiment 3: Subculture of prawn 3D culture cells

[0046] Taking the subculture of shrimp hemolymphocytes inoculated and grown on the surface of Matrigel as an example, the specific steps are as follows:

[0047] (1) Wash the cells once with pre-cooled PBS solution on ice;

[0048] (2) Add the improved cell recovery solution (Corning, Cat.NO.354253) at a ratio of 2 mL / petri dish (φ35mm); place the petri dish on ice and shake it left and right until the Matrigel dissolves;

[0049] (3) Collect the dissolved Matrigel-cell suspension into a 1.5mL centrifuge tube, centrifuge at 3000g, 4°C for 5-10 minutes, and discard the supernatant;

[0050] (4) Resuspend the cell pellet in the complete culture medium of prawn cells, inoculate it on Matrigel in a new culture plate, and store it at 28°C in 3% CO 2 cultured in an incubator.

[0051] The preparation method of the above PBS solution: weigh 12.0g NaCl, 0.3g KCl, 4.5g NaCl respectively 2 HPO 4 12H 2 O and 0.3 g of KH 2...

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Abstract

The invention provides a continuous culture technology of prawn cells by establishing an effective prawn cell 3D culture and subculture method. The continuous culture technology can be applied to establishment of prawn immortal cell lines. The addition ratio of matrigel is optimized, and thus, a preparation method of the matrigel for 3D culture of the prawn cells is provided. A formula of a complete culture medium of the prawn cells is further optimized, the complete culture medium of the prawn cells is selected as an anticoagulant and a diluent of prawn blood lymphocytes, a 3D culture mode of attached growth on the surface of the matrigel is selected, thus, the separation and 3D culture technology of the prawn peripheral blood lymphocytes is provided, the prawn peripheral blood lymphocytes are attached to the surface of the matrigel for growth by a mode of round single cell and cell clusters/balls, and the survival and growth abilities of the prawn peripheral blood lymphocytes are higher than those of the prawn peripheral blood lymphocytes subjected to 2D culture. The invention also provides a passaging method for culturing the prawn cells in a 3D mode, the passaged prawn blood lymphocytes can be well attached to the surface of the matrigel for growth, and the survival rate of the passaged prawn blood lymphocytes can reach up to 90% or above.

Description

technical field [0001] The invention belongs to the technical field of animal cell and tissue culture, and in particular relates to a method for continuous culture of prawn cells. Background technique [0002] The frequent outbreak of shrimp virus disease has brought a heavy blow to the shrimp farming industry, and has become a bottleneck problem restricting the sustainable development of the shrimp farming industry. The establishment of immortalized shrimp cell lines can provide effective research means and carriers for the isolation and purification of shrimp viruses, the study of their pathogenic mechanisms, and the production of highly effective antiviral vaccines. Although experts and scholars at home and abroad have made a lot of explorations and efforts on the establishment of prawn cell lines, because the prawn cells hardly divide under the established culture system, and the prawn cells are extremely resistant to various proteases including trypsin. Sensitive, so t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2500/84C12N2501/11C12N2501/115C12N2513/00C12N2500/32C12N2500/34C12N2500/82C12N2500/30C12N2500/60
Inventor 郭华荣宋欣周阳
Owner OCEAN UNIV OF CHINA
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