Technology for dissociating and culturing prawn embryonic cells

A technology of embryonic cells and culture methods, which is applied in the field of aseptic isolation and in vitro culture of prawn embryonic cells, can solve the problems of no successful reports of line establishment, no continuous monolayer formation, and inability to obtain living cells, etc., and achieve operational Simple and fast, fast adherence to the wall, not easy to pollute the effect

Active Publication Date: 2016-07-13
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Frerichs used M. rosenbergii 7-13-day embryos as materials, and used M199 medium and grinding method to successfully obtain primary cultured embryonic cells that adhered to the wall, but did not form a continuous monolayer, and the cells did not grow after mechanical passage. reattachment
Six years later, Fan Tingjun et al. (2002) used MPS medium supplemented with growth factors (bFGF and IGF-II) and the grinding method to obtain a continuous monolayer of embryonic cells of Penaeus sinensis, but there was no report on the success of the subsequent line establishment. , and the results have not b...

Method used

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  • Technology for dissociating and culturing prawn embryonic cells
  • Technology for dissociating and culturing prawn embryonic cells
  • Technology for dissociating and culturing prawn embryonic cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Cleaning and disinfection treatment technology of early stage embryos of Penaeus japonicus.

[0023] Specific steps are as follows:

[0024] (1) Preparation of sterilized seawater: Fresh natural seawater was filtered through 16-20 layers of gauze and a 0.22 μm filter membrane in sequence, sterilized by high-pressure steam for 20 minutes, and cooled for later use.

[0025] (2) Materials for early embryos of prawns: Collect embryos at the limb bud stage 6-8 hours after spawning from the prawn nursery. box and transported back to the laboratory.

[0026] (3) Collection of prawn embryos: let it stand for a few minutes, and after the shrimp eggs settle to the bottom, use a plastic straw to collect the prawn embryos into a 50mL centrifuge tube, wash them with sterilized seawater for 5-6 times, and the suspension will die during the washing process. Embryos and impurity particles are sucked out.

[0027] (4) Washing of shrimp embryos: transfer the embryos to a 70-...

Embodiment 2

[0032] Example 2 Screening of the best dissociation method for the embryonic cells of Penaeus japonicus.

[0033] The outer layer of the embryo of the prawn has an egg shell formed by the lifting of the fertilization membrane to protect the embryo from the damage of the external environment. In order to obtain dispersed early embryonic cells, it is necessary to break the outer elastic and tough egg shell. Since the fertilized eggs of prawns are homoyolk eggs with complete cleavage, it is difficult to separate and culture early embryonic cells. The present invention has compared the separation and culture effect of 4 kinds of physical dissociation methods, as attached Figure 2-3 As shown, it is found that the separation effect of the stainless steel filter mashing method is the best, and a large number of living and complete embryonic cells and cell clusters can be obtained.

[0034] (1) Stainless steel filter smashing method: transfer the aseptically treated prawn embryos i...

Embodiment 3

[0040] Example 3 Culture method of Penaeus japonicus embryonic cells.

[0041] Specific steps: Inoculate the cell suspension of prawn embryos into culture flasks or culture plates, and inoculate at 28°C, 3% CO 2 cultured in an incubator. Observe the cell adherent growth every day, and replace 1 / 2 of the medium every 2 days according to the color change of the medium.

[0042] as attached Figure 4 As shown, the embryonic cells and cell clusters separated by the stainless steel filter smashing method can adhere to the wall and grow 2-3 hours after inoculation. The cells become elongated and spread out. There is a clear growth trend. After 48 hours, the adherent cells in the adjacent large cell clusters contacted each other and reached a semi-converged state. At this time, the amount of yolk granules decreased significantly.

[0043] The complete culture medium for prawns provided by the present invention optimizes whether to add the prawn muscle extract and the addition amo...

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Abstract

The invention provides a technology for dissociating and culturing prawn embryonic cells effectively, and applies the technology to research of prawn embryonic cell line establishment. According to the technology, the conventional sterile disinfection treatment technology for prawn embryos is optimized, that is, prawn eggs can be collected from a prawn breeding plant directly and transported to a laboratory for treatment; particularly, as iodophor treatment concentration is optimized, not only can pollution of microorganisms and protozoa be controlled effectively, but also toxicity of iodophor to the prawn embryos is reduced to the most extent; the formula of a prawn complete medium is optimized to provide a properer nutritional condition for adherence and growth of the prawn embryonic cells, so that adherence is faster, and growth is better; moreover, the formula of the medium is simplified, so as to reduce the cost of the medium; an embryonic cell collector made of a stainless-steel filter screen of a proper aperture is used for mashing the prawn embryos, so that a large quantity of living prawn embryos and cell clusters can be obtained in a short time; the operation is simple, convenient and fast, and lasts for only several seconds; pollution is reduced; the effect is far better than those of the other reported dissociating methods.

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 aseptic separation and in vitro culture of prawn embryo 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 or drugs. Although experts and scholars at home and abroad have made a lot of exploration and efforts on the establishment of prawn cell lines, the immortalized cell lines have not been successfully established because the in vitro cultured cells derived from adult tissue of prawns d...

Claims

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Application Information

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IPC IPC(8): C12N5/07
CPCC12N5/0601C12N2509/00
Inventor 郭华荣张晓娟陈月如
Owner OCEAN UNIV OF CHINA
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