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In vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells, and in vitro culture and cryopreservation method

A technology of channel catfish and spermatogonial stem cells, which is applied in the direction of cell culture active agents, germ cells, animal cells, etc., can solve the problems of complex composition of SSCs medium formula and short culture time of SSCs in vitro, and achieve easy acquisition and preparation, long-term Stable in vitro culture, low cost effect

Inactive Publication Date: 2017-03-22
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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Problems solved by technology

[0004] The purpose of the present invention is to solve the problem that the existing SSCs in vitro culture time is relatively short, and the composition of the SSCs medium formula is complicated, and provide a kind of in vitro culture medium, cryopreservation liquid and in vitro culture and cryopreservation of channel catfish spermatogonial stem cells method

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  • In vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells, and in vitro culture and cryopreservation method
  • In vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells, and in vitro culture and cryopreservation method
  • In vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells, and in vitro culture and cryopreservation method

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specific Embodiment approach 1

[0019] Specific embodiment 1: In this embodiment, the in vitro culture solution of channel catfish spermatogonial stem cells is based on L-15 medium, and 25 μmol of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 100 units of penicillin, and 100 μg of chain are added per milliliter. Mycin, 1.0 μg NaHCO 3 , 0.3μg L-glutamic acid, 0.2mL fetal bovine serum (FBS), 0.05mL catfish serum, 1ng fibroblast growth factor (bFGF), the pH value is 7.2-7.4.

[0020] In this embodiment, a 1 mol / L HCl solution or a 1 mol / L NaOH solution is used to adjust the pH value.

[0021] The in vitro culture solution of channel catfish spermatogonial stem cells configured in this embodiment is sterilized by filtration with a 0.22 μm filter membrane, subpackaged, and stored at 4°C.

specific Embodiment approach 2

[0022] Specific embodiment two: the in vitro culture method of channel catfish spermatogonial stem cells in this embodiment is carried out according to the following steps:

[0023] 1. Channel catfish testis cell isolation and SSCs enrichment;

[0024] 2. Cultivation of SSCs: inoculate the SSCs enriched in step 1 onto a cell culture plate covered with gelatin, and place them in an incubator with saturated humidity and a constant temperature of 26°C in an atmospheric environment; wherein, the cultivation of SSCs is selected for specific implementation The in vitro culture solution of channel catfish spermatogonial stem cells described in way one.

[0025] In step 1 of this embodiment, channel catfish testis cell isolation and SSCs enrichment can be carried out as follows: Second-year-old channel catfish (full length 25-30 cm) that have not reached sexual maturity are anesthetized with MD-222, placed on ice and brought back laboratory. After the body surface of each fish was d...

specific Embodiment approach 3

[0040] Specific implementation mode three: the difference between this implementation mode and specific implementation mode two is: in step two, press 8×10 6 ~10×10 6 Inoculate the SSCs enriched in Step 1 onto a cell culture plate lined with 0.1% gelatin at a density of 0.1 / mL. Other steps and parameters are the same as those in Embodiment 2.

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Abstract

The invention relates to an in vitro culture solution and cryopreservation solution of fish spermatogonial stem cells (SSCs), and an in vitro culture and cryopreservation method, and solves the existing problems of relatively short in vitro culture time of SSCs and complex components of SSCs medium. The in vitro culture solution is prepared by taking an L-15 medium as the basis and adding HEPES, penicillin, streptomycin, NaHCO3, L-glutamic acid, fetal bovine serum, catfish serum and bFGF. The in vitro culture method includes: 1. Ietalurus Punetaus testis cell separation and SSCs enrichment; and 2. SSCs culture. The in vitro culture solution and cryopreservation solution of Ietalurus Punetaus spermatogonial stem cells provided by the invention have the characteristics of simple formula components, easy acquisition and preparation, and low price, and can realize long-term in vitro culture and cryopreservation of Ietalurus Punetaus spermatogonial stem cells.

Description

technical field [0001] The invention relates to an in vitro culture medium, a cryopreservation solution and an in vitro culture and cryopreservation method for fish spermatogonial stem cells. Background technique [0002] Fish spermatogonial stem cells (SSCs) are a kind of reproductive stem cells that can self-renew and differentiate into sperm in the gonads of male fish. The in vitro culture of fish SSCs is of great significance for the study of the occurrence, development, differentiation and regulation mechanism of animal reproductive stem cells. SSCs in vitro culture technology combined with SSCs allografting technology can be used to shorten the reproductive cycle of important economic fish species and save endangered fish species; SSCs cultured in vitro can also be used for fish virology and toxicology research. [0003] The in vitro isolation and culture of SSCs has been reported in mice, rats, humans, pigs and other mammals, but there are few studies on fish SSCs. ...

Claims

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Application Information

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IPC IPC(8): C12N5/076A01N1/02
CPCC12N5/061A01N1/0252C12N2500/12C12N2500/30C12N2500/32C12N2501/113C12N2501/115C12N2501/119
Inventor 尚梅苏宝锋
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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