Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle

A technology for primary culture and embryonic cells, applied in the field of cell culture, can solve the problems of low culture success rate, harsh conditions, complicated operation, etc., and achieve the effect of high culture success rate, simple operation and convenient culture.

Active Publication Date: 2014-09-24
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method has the disadvantages of complex operation, harsh conditions and low success rate of cultivation

Method used

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  • Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle
  • Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle
  • Primary culture method of embryonic cell tissue pieces of Chinese softshell turtle

Examples

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Embodiment 1

[0028] A method for primary culture of soft-shelled turtle embryo cell tissue block, comprising the following steps:

[0029] (1) Fertilized eggs hatched for 15 days were collected, and the surface of the fertilized eggs was sprayed with 70% alcohol for disinfection.

[0030] (2) Then use ophthalmic scissors to cut the eggshell, use ophthalmic curved tweezers to peel off the embryo from the egg yolk, put the stripped embryonic tissue in 1×PBS buffer (PH 7.4, add 500U / ml penicillin and 500U / ml streptomycin), and then sterilized by immersing in 70% alcohol for the first time for 15 seconds, and then sterilizing by immersing in another 70% alcohol for the second time for 15 seconds.

[0031] (3) Put the embryonic tissue into M199 medium (500U / ml penicillin and 500U / ml streptomycin are added to M199 medium), wash 5 times, and then transfer to complete medium (complete medium is M199 medium Add 100U / ml penicillin, 100U / ml streptomycin, 20% fetal bovine serum and 2mmol / L glutamine)...

Embodiment 2

[0034] A method for primary culture of soft-shelled turtle embryo cell tissue block, comprising the following steps:

[0035] (1) Fertilized eggs hatched for 25 days were collected, and the surface of the fertilized eggs was sprayed with 75% alcohol for disinfection.

[0036] (2) Then use ophthalmic scissors to cut the eggshell, use ophthalmic curved tweezers to peel off the embryo from the egg yolk, put the stripped embryonic tissue in 1×PBS buffer (PH 7.4, add 500U / ml penicillin and 500U / ml streptomycin), and then sterilized by immersing in 75% alcohol for the first time for 10 seconds, and then sterilizing by immersing in another 75% alcohol for 10 seconds for the second time.

[0037] (3) Put the embryonic tissue into M199 medium (500U / ml penicillin and 500U / ml streptomycin are added to M199 medium), wash 4 times, and then transfer to complete medium (complete medium is M199 medium Add 100U / ml penicillin, 100U / ml streptomycin, 20% fetal bovine serum and 2mmol / L glutamine)...

Embodiment 3

[0040] A method for primary culture of soft-shelled turtle embryo cell tissue block, comprising the following steps:

[0041] (1) Collect fertilized eggs hatched for 19 days (such as figure 1 shown), the surface of fertilized eggs was sprayed with 75% alcohol to disinfect.

[0042] (2) Then use ophthalmic scissors to cut the eggshell (such as figure 2 Shown), use ophthalmic curved forceps to peel off the embryo from the egg yolk, put the stripped embryonic tissue in 1×PBS buffer (PH 7.4, add 500U / ml penicillin and 500U / ml streptomycin) Wash twice in 75% alcohol for the first time, then soak for 12 seconds in 75% alcohol for the first time, and then sterilize for 12 seconds in another 75% alcohol for the second time.

[0043] (3) Put the embryonic tissue into M199 medium (500U / ml penicillin and 500U / ml streptomycin are added to M199 medium), wash 4 times, and then transfer to complete medium (complete medium is M199 medium Add 100U / ml penicillin, 100U / ml streptomycin, 20% f...

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Abstract

The invention discloses a primary culture method of the embryonic cell tissue pieces of a Chinese softshell turtle. The primary culture method comprises the following steps of: (1) collecting the fertile eggs of the Chinese softshell turtle, and disinfecting; (2) breaking the eggshells of the fertile eggs, separating embryos from yolk, cleaning the separated embryonic tissues with a 1*PBS (Phosphate Buffer Solution), and then soaking for sterilization; (3) cleaning the embryonic tissues for 4-5 times in an M199 culture medium, then transferring into a complete culture medium, cutting the embryonic tissues into pieces to obtain tissue pieces, and then cleaning the tissue pieces once by using the complete culture medium; (4) uniformly dispersing the tissue pieces to the inner wall of a cell bottle by using a sterile disposable inoculating needle so that the tissue pieces grow for 3-4 hours in a way of being attached to the wall, then adding the complete culture medium to submerge the tissue pieces, and culturing at the constant temperature of 30-31 DEG C for more than 3 days. The primary culture method disclosed by the invention has the advantages of easy operation, convenient culture, no need of specific cell separation liquid and high culture success rate.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for primary culture of soft-shelled turtle embryo cell tissue block. Background technique [0002] Chinese soft-shelled turtle, also known as water fish, soft-shelled turtle, and turtle, is a common farmed turtle species. Soft-shelled soft-shelled turtles live in rivers, lakes, ponds, reservoirs and other freshwater waters with gentle currents and abundant fish and shrimp, and often appear in Dashan streams. Activities are more frequent on quiet, clean, sunny water shores, and sometimes go ashore but not too far away from the water source. It can crawl and climb on land and swim freely in water. Like to bask in the sun or enjoy the cool wind. Folk proverbs describe the activities of soft-shelled turtles as "walking up the beach in spring, living in the shade of willows in the scorching summer, entering the bottom of the water when it is cold in autumn, and drilling into th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
Inventor 曹铮刘莉林锋叶雪平
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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