Vitrifiation freezing method for chicken embryo primordial germ cell

A primordial germ cell, vitrification freezing technology, applied in biochemical equipment and methods, microorganisms, preservation of human or animal bodies, etc., can solve problems such as inconvenient operation, and achieve the effect of simple operation

Inactive Publication Date: 2006-08-23
YANGZHOU UNIV
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Problems solved by technology

At present, the slow freezing method to preserve chicken embryo PGCs cells has been successful, but the slow free

Method used

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  • Vitrifiation freezing method for chicken embryo primordial germ cell
  • Vitrifiation freezing method for chicken embryo primordial germ cell
  • Vitrifiation freezing method for chicken embryo primordial germ cell

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Experimental program
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Embodiment Construction

[0014] Step 1: Isolation and extraction of chicken embryo PGCs

[0015] PGCs were isolated at the 19th (68-72h) and 28th (5.5d) phases of the two aggregation peaks of PGCs. Fertilized eggs hatched to the 19th stage (68-72 hours of incubation), the chicken embryos were aseptically collected, washed 3 times with calcium and magnesium-free fetal bovine serum (PBS), the head and tail were removed under a dissecting microscope, and the tissue pieces of the gonads were obtained. Wash with PBS for 3 times, transfer to a Erlenmeyer flask, digest with Trypsin-EDTA cell digestion solution at room temperature for 3-4min, and blow gently with a glass tube to disperse during digestion. The digestion was terminated with 10% FCS of the amount of the digestion solution, filtered through a 100-mesh sieve, and the cells were collected by centrifugation at 180 g for 8 min. The precipitate was resuspended with 0.1 mL of TCM-199 culture medium without calf serum, transferred into a finger tube (2...

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Abstract

The vitrification freezing method of primordial chicken embryo germ cell is to vitrify and freeze primordial germ cell of chicken embryo sex gland in the 19th stage and 28th stage of growth. After it is added into vitrifying and freezing liquid, primordial chicken embryo germ cell is transferred into freezing tube, balanced at 4 +/-1 deg.c for 5 min, stilled on surface of liquid nitrogen for 0.5-1.5 min and set directly into liquid nitrogen. The present invention has simple and quick operation, and stable and reliable preservation effect, and may be used in preserving various traditional bird resources.

Description

technical field [0001] The invention relates to a method for vitrification and cryopreservation of gonad primordial germ cells developed to the 19th stage (about 3 days after hatching) and the 28th stage (about 5.5 days after hatching). Background technique [0002] Vitrification is an ultra-fast freezing method first proposed by Rall and Fahy in 1985. The basic principle of vitrification is to add a high concentration of cryoprotectant during the freezing process of cells, and put them directly into liquid nitrogen after solidification and dehydration. Through rapid cooling, a glassy state is formed to avoid ice crystal damage inside and outside the cells, and the glassy state is preserved at low temperature. Although vitrification started late in cryopreservation technology and cryobiological research, it has attracted much attention due to its good freezing effect and potential development prospects, and has made rapid progress. So far, vitrification cryopreservation has...

Claims

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Application Information

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IPC IPC(8): C12N5/06A01N1/02
Inventor 李碧春韩威
Owner YANGZHOU UNIV
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