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Sturgeon embryonic cell line, culture medium and preparation method of culture medium

A technology of embryonic cells and culture methods, applied in the culture medium and its preparation, and the field of sturgeon embryonic cell lines, which can solve the problems of difficult establishment of sturgeon embryonic cell lines, extremely difficult to distinguish embryo body and yolk, and inability to transfer embryos, etc. Achieve the effect of vigorous proliferation in vitro, easy operation, and good growth and division state

Active Publication Date: 2022-03-01
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the experiment, we found that the yolk and the embryo are closely connected, it is extremely difficult to distinguish the embryo body from the yolk, and the embryo body is very fragile, and the embryo cannot be transferred with tools
Therefore, sturgeon embryonic cell lines are difficult to establish

Method used

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  • Sturgeon embryonic cell line, culture medium and preparation method of culture medium
  • Sturgeon embryonic cell line, culture medium and preparation method of culture medium
  • Sturgeon embryonic cell line, culture medium and preparation method of culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Establishment of Yangtze River Acipenser Embryo Cell Line

[0041] Follow the steps below to establish the Changjiang sturgeon embryonic cell line

[0042](1) Removal of egg membrane and egg shell: collect 15 fertilized eggs of Yangtze River sturgeon hatched for 48-96 hours, add 20ml PBS to wash 4 times, then transfer to a cell culture dish, use ophthalmic surgical forceps to peel off the outer layer of egg membrane, and then wash with 75 The egg shells (including the contents) were washed with % alcohol for 4 times, and then washed with sterile PBS for 4 times. Transfer the non-coated sturgeon eggs to a new sterile petri dish, use ophthalmic surgical forceps to peel off the egg shells, and release the contents;

[0043] (2) Separation of embryo body and yolk: Add 5ml of antibiotic-containing PBS washing solution to the container containing the embryo body and yolk in the previous step, and set the rotation speed through a horizontal shaker (or a hand-held p...

Embodiment 2

[0054] Implementation 2: Morphological Observation of Changjiang Acipenser Embryo Cell Line

[0055] The morphology of the primary cells and the 17th passage cells was compared and observed by an inverted microscope. Cells with a confluence of 80% to 90% were taken for observation, primary cells ( figure 1 ) contains cells of various shapes, most of which are fibrous and polygonal. 17 passage cells ( figure 2 ) was the same as the primary one, and the cells were mainly fibroblasts and polygonal cells.

Embodiment 3

[0056] Embodiment 3: verification of resuscitated vitality of Changjiang sturgeon embryonic cell line

[0057] (1) Take out the frozen Yangtze River sturgeon embryo cells from liquid nitrogen, and shake them quickly in warm water at 37°C to melt the cells as soon as possible. cell.

[0058] (2) Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, add culture medium to resuspend the cells, stain with trypan blue, and count using an inverted microscope. Cells stained blue are dead cells, and cells that are not stained are live cells. Cell viability was calculated after counting. The results in Table 1 show that the viability of the cells after recovery was above 70%.

[0059] Table 1 Comparison of the viability of different generations of cells before cryopreservation and after recovery

[0060] Frozen cell passage Viability before cryopreservation post-resuscitative vitality P5 98％ 75％ P12 97％ 81％ P15 98％ 77％ P17 98％ 84％ ...

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Abstract

The invention relates to a sturgeon embryonic cell line, a culture medium and a preparation method of the sturgeon embryonic cell line, and belongs to the technical field of cell biology, the sturgeon embryonic cell line ADEc strain is preserved in the China General Microbiological Culture Collection Center on July 28, 2021, and the preservation number is CGMCC NO.22360. The invention also provides a sturgeon embryonic cell line culture medium, and the key step of preparing the sturgeon embryonic cell line by using the culture medium is to separate embryos and yolk by adopting horizontal oscillation, and collect pure and complete sturgeon embryonic cells. According to the method, the sturgeon embryonic cell line is successfully obtained, the morphological characteristics of primary cells are still kept after continuous passage for 20 generations, and the cells still can keep high activity after cryopreservation and recovery.

Description

technical field [0001] The invention belongs to the technical field of cell biology, in particular to a sturgeon embryo cell line, a culture medium and a preparation method thereof. Background technique [0002] Yangtze River sturgeon (Acipenserdabryanus), also known as Dabry's sturgeon, belongs to the genus Acipenseridae of the order Acipenseridae. It is mainly distributed in the upper reaches of the Yangtze River. It is another species of sturgeon in the Yangtze River in my country except the Chinese sturgeon. It is a critically endangered species by the International Union for Conservation of Nature. In recent years, due to environmental changes and human activities, the survival of the Yangtze River sturgeon has been severely challenged, and the natural population has declined severely. Although the relevant departments adopt methods such as in-situ conservation and ex-situ conservation for the protection of genetic resources, due to limited conditions, problems such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12R1/91
CPCC12N5/0603C12N2501/115C12N2501/11C12N2500/44C12N2500/84Y02A40/81
Inventor 巩志宏王娜陈松林刘娟娟张德志杜合军
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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