Antheraea pernyi ovary cell culture medium and application thereof

An ovarian cell and cell culture technology, applied in the fields of insect anatomy and insect tissue culture, can solve the problems of silkworm farmers reducing production, affecting the enthusiasm of tussah silkworm, and failing to harvest, and achieves the effect of convenient operation and comprehensive cell growth nutrients.

Active Publication Date: 2012-08-01
辽宁省农业科学院大连生物技术研究所
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AI Technical Summary

Problems solved by technology

However, because tussah silkworms are kept in the wild, they are subjected to the invasion of various pathogenic microorganisms such as viruses, bacteria, and microsporidia, and diseases occur from time to time, resulting in reduced or even no harvest for some silkworm farmers. The development of tussah silkworm production, and tussah silkworm cells are the necessary tools and platforms to study the pathogenesis of these diseases and the law of pathogenic infection. Therefore, it is of great significance to carry out research on tussah silkworm cell culture methods and establish tussah silkworm ovary cell culture methods for the development of tussah silkworm production and related research

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  • Antheraea pernyi ovary cell culture medium and application thereof
  • Antheraea pernyi ovary cell culture medium and application thereof
  • Antheraea pernyi ovary cell culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 tussah ovary cell medium MLM-45 formula composition and preparation method

[0041] The composition of the medium formula and the preparation of the culture medium. The culture medium consists of amino acids, sugars, inorganic salts, vitamins and cell growth-promoting additives.

[0042] Table 1. Prepare 5 times 1000mL amino acid and sugar mixed storage solution (×5, MLM-AAS), its composition and dosage:

[0043] 5x MLM-AAS ingredients Dosage (g) L-Cystine (L-cystine) 0.28 L-Tyrosine (L-Tyrosine) 0.425 α-Alanine (α-alanine) 1.55 β-Alanine (β-alanine) 1.05 L-Arginine-HCl (L-Arginine) 3.45 L-Aspartic acid (L-Aspartic Acid) 3.75 L-Asparagine (L Asparagine)- 3.75 L-Glutamic acid (L-glutamic acid) 4.5

[0044] L-Glutamine (L-Glutamine) 4.5 L-Glycine (L-Glycine) 2.3 L-Histidine (L-Histidine) 4.25 L-Isoleucine (L-Isoleucine) 2.6 L-Leucine (L-le...

Embodiment 2

[0056] Embodiment 2 tussah ovary cell culture method in vitro

[0057] The materials of tussah silkworm variety Qing No. 6 (5th instar larvae and pupae) were provided by Liaoning Sericulture Research Institute.

[0058] 1. The acquisition of tussah ovary cells:

[0059] (a) Acquisition of tussah silkworm chrysalis ovarian cells: take healthy tussah silkworm chrysalis, sterilize the body surface with 75% ethanol for 20 minutes, wash 3 times with sterile distilled water in an ultra-clean workbench, blot the body surface moisture with sterile gauze, and use a pin Fix in a dissecting wax dish sterilized by UV for 2h. Aseptically cut open the abdomen, dissect with ophthalmic scissors and tweezers, take out the ovarian tissue in sterile Corlson buffer solution (1000ml Corlson solution formula: NaCl 7g, KCl 0.2g, CaCl 2 0.2g, MgCl 2 .6H2O 0.1g, NaH 2 PO4 0.2g, NaHCO 3 0.05 g, glucose 8 g) in a small plate, remove the adipose tissue and capsule etc. on the ovary, then transfer ...

Embodiment 3

[0064] Embodiment 3 tussah ovary cell culture conditions in vitro

[0065] Utilize existing commercial insect culture medium (MM, TC-100, MGM-443, GRACE, IPL-41, MGM-448, TNM-FH, SF-900) and the MLM-45 culture that embodiment 1 makes Based on cell growth experiments.

[0066] Select the above-mentioned different insect culture medium, culture tussah silkworm chrysalis and larval ovarian cells under the same culture conditions for 2 months, observe under the phase-contrast inverted microscope and find that the growth of MLM-45 medium cells is better than that of other kinds, count by Trypan Blue staining Cell viability (see Table 5). MLM-45 medium was used to conduct cell growth experiments at different temperatures (4°C, 15°C, 20°C, 26°C, 27°C, 28°C, 30°C, 37°C) to determine the optimal growth temperature of cells (Table 6 ). MLM-45 medium was used to test the effect of different pH (pH5.5, 6.0, 6.2, 6.3, 6.4, 6.5, 7.0) on cell growth, and the optimum cell growth pH was det...

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Abstract

The present invention discloses composing components of an antheraea pernyi ovary cell culture medium, a preparation method of the antheraea pernyi ovary cell culture medium, and an application of the antheraea pernyi ovary cell culture medium in antheraea pernyi ovary cell culture. According to the present invention, a prepared amino acid and sugar mixing storage solution (MLM-AAS), an inorganicsalt storage solution (MLM-Salt), a vitamin storage solution (MLM-Vit), bovine serum albumin, fetuin and the like are prepared according to a certain ratio, and the pH value is adjusted to 6.2-6.4 toobtain a MLM-45 culture medium, wherein the prepared MLM-45 culture medium is added with fetal bovine serum and a penicillin and streptomycin mixing solution according to a certain volume ratio before the prepared MLM-45 culture medium is used, the volume ratio of the fetal bovine serum is 20%, and the volume ratio of the penicillin and streptomycin mixing solution is 1%. With a plurality of experiments, the following in vitro cell growth conditions are determined that: the culture temperature is 26-28 DEG C, the pH value is 6.2-6.4, and the osmotic pressure is 315-350 mOsm / kg. With the present invention, technical services are provided for researches in antheraea pernyi biology, antheraea pernyi pathogenic microorganism and related fields.

Description

technical field [0001] The invention relates to the fields of insect anatomy, insect tissue culture and the like, in particular to a tussah ovary cell culture medium and its application in the in vitro culture technology of tussah ovary cells. Background technique [0002] Insect cell culture is a technology that removes cells from insects, simulates the physiological environment of insects, and enables cells to survive, grow, and maintain structure and function under sterile, suitable, and rich nutrient conditions. Because primary cells have just been separated from the tissue, the biological characteristics have not changed much, and they still retain the original genetic characteristics, and are the closest to and reflect the growth characteristics in vivo. They are suitable for experimental research such as drug sensitivity tests and cell differentiation. In the early 20th century, many entomologists tried to use insect cells cultured in vitro as tools for scientific res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 王林美李树英岳冬梅范琦
Owner 辽宁省农业科学院大连生物技术研究所
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