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Separation and in-vitro culture method for pelodiscus sinensis ovary cells

A technology for ovarian cells and in vitro culture, which is applied in the field of ovarian cell separation and in vitro culture, can solve the problems of limited, hindered species proliferation and release, and resource restoration, and achieves the effects of strong repeatability, novel steps, and high survival rate

Inactive Publication Date: 2017-06-13
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, domestic and foreign research on germplasm preservation and cell engineering breeding technology of these species is still extremely limited, which seriously hinders the development of research work on the proliferation and release of these species and resource restoration.

Method used

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  • Separation and in-vitro culture method for pelodiscus sinensis ovary cells
  • Separation and in-vitro culture method for pelodiscus sinensis ovary cells
  • Separation and in-vitro culture method for pelodiscus sinensis ovary cells

Examples

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Effect test

Embodiment 1

[0028] Such as figure 1 As shown, the Chinese soft-shelled turtle ovary cell isolation and in vitro culture method provided in the present embodiment comprises the following steps:

[0029] (1) Select sexually immature 1-year-old female soft-shelled turtles, ether anesthetize bloodletting, 70% (volume percentage content) alcohol spray sterilization, dissect and take out the ovarian tissue, the ovarian tissue is washed with phosphate buffer solution and cut into pieces, put into test tube;

[0030] (2) Digest the ovarian tissue of soft-shelled turtle with collagenase and trypsin successively, and then collect the cells by centrifugation;

[0031] (3) Put the cells collected by centrifugation into a treated cell culture bottle and add complete medium for culturing;

[0032] (4) After the cells proliferate and cover the bottom of the bottle, they are digested with trypsin and subcultured or cryopreserved to obtain Chinese soft-shelled turtle ovary cells.

[0033] In the above ...

Embodiment 2

[0041] Such as figure 1 As shown, the Chinese soft-shelled turtle ovary cell isolation and in vitro culture method provided in the present embodiment comprises the following steps:

[0042] (1) Select sexually immature 1-year-old female soft-shelled turtles, ether anesthetize for bloodletting, 80% (volume percentage) alcohol spray sterilization, dissect and take out the ovarian tissue, wash the ovarian tissue with phosphate buffer solution and cut it into pieces, put it into a test tube ;

[0043] (2) Using collagenase and trypsin to digest the ovarian tissue of soft-shelled turtle successively, and then centrifuge to collect the cells;

[0044] (3) Put the cells collected by centrifugation into a treated cell culture bottle and add complete medium for culturing;

[0045] (4) After the cells proliferate and cover the bottom of the bottle, they are digested with trypsin and subcultured or cryopreserved to obtain Chinese soft-shelled turtle ovary cells.

[0046] In the above ...

Embodiment 3

[0053] Such as figure 1 As shown, the Chinese soft-shelled turtle ovary cell isolation and in vitro culture method provided in the present embodiment comprises the following steps:

[0054] (1) Select sexually immature 2-year-old female soft-shelled turtles, ether anesthetize for bloodletting, 90% (volume percentage) alcohol spray sterilization, dissect and take out the ovarian tissue, wash the ovarian tissue with phosphate buffer solution and cut it into pieces, put it into a test tube ;

[0055] (2) Using collagenase and trypsin to digest the ovarian tissue of soft-shelled turtle successively, and then centrifuge to collect the cells;

[0056] (3) Put the cells collected by centrifugation into a treated cell culture bottle and add complete medium for culturing;

[0057] (4) After the cells proliferate and cover the bottom of the bottle, they are digested with trypsin and subcultured or cryopreserved to obtain Chinese soft-shelled turtle ovary cells.

[0058] In the above ...

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Abstract

The invention discloses a separation and in-vitro culture method for pelodiscus sinensis ovary cells. The separation and in-vitro culture method comprises the following steps: (1) selecting sexually immature female pelodiscus sinensis; sterilizing and taking out in-vitro ovary tissues; washing the in-vitro ovary tissues with a phosphate buffering solution and washing; putting the in-vitro ovary tissues into a container; (2) carrying out digestion treatment on the washed in-vitro ovary tissues through adopting collagenase and pancreatin, and centrifuging and collecting the cells; (3) putting the collected cells into a treated cell culture container and adding a complete culture medium for culturing; (4) after the cells are proliferated and fully spread on the bottom of the cell culture container, digesting by utilizing the pancreatin and carrying out subculture or freezing preservation to obtain the pelodiscus sinensis ovary cells. The method disclosed by the invention has novel steps, is simple and convenient and has high repeatability; a pelodiscus sinensis ovary cell line with a high survival rate can be obtained.

Description

technical field [0001] The invention belongs to the technical field of ovarian cell separation and in vitro culture, in particular to a method for the separation and in vitro culture of soft-shelled turtle ovary cells. Background technique [0002] So far, domestic researchers have tried cell culture of various body tissues of turtles and turtles, including embryonic primary cell culture, turtle brain, liver, spleen, kidney and heart tissue cells. Among them, except for the fibroblast culture of heart tissue, a stable cell line was obtained, and the basic culture of other tissue cells showed "spontaneous transformation" phenomenon or decline after only 10 passages, and the cells could not continue to be subcultured. Even the heart fiber cell line that has obtained a stable cell line grows slowly compared with other animal cell lines, and has a strong dependence on temperature and passage time. The inability to obtain stable turtle cell lines with good growth is mainly due t...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0682
Inventor 徐红艳朱新平李伟
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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