A kind of separation and culture method of prawn embryo cells

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 specifically relates to a method for aseptic separation and in vitro culture of shrimp embryo cells. Background technique [0002] Frequent outbreaks of prawn virus diseases have dealt a heavy blow to the shrimp aquaculture industry and become a bottleneck restricting the sustainable development of the shrimp aquaculture industry. The establishment of shrimp immortal cell lines can provide effective research methods and carriers for the isolation and purification of shrimp viruses, the study of its pathogenic mechanism, and the production of efficient antiviral vaccines or drugs. Although domestic and foreign experts and scholars have made a lot of exploration and efforts on the establishment of shrimp cell lines, the immortalized cell line has not been successfully established because the in vitro cultured cells derived from adult shrimp tissues do not divide and are diffic...

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 been replicated by others

Seven years later, Yu Liming et al. (2009) explored the dissociation and culture methods of Chinese prawn blastocysts and gastrula cells, and found that the dissection method is suitable for isolating blastocyst cells, and the hemocytometer plate pressing method is suitable for isolating gastrula Blast cells, the cultured monolayer of embryonic cells can be maintained in vitro for 1-5 weeks, but no obvious proliferation

The dissecting and tableting methods are inefficient and prone to contamination, while the grinding, sieving and needle methods cannot obtain enough viable cells

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