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Hexagrammos otakii spermatogonium culture medium and culture method

A technology of six line fish and spermatogonia is applied in the field of cell biology, which can solve the problems of low egg production and difficulty in artificial breeding, and achieve the effect of promoting proliferation and facilitating collection.

Inactive Publication Date: 2021-08-24
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the small amount of eggs laid by Otaki six-line fish, and about 20 minutes after the eggs are released, the eggs start to become sticky, and the eggs stick to each other and cling to the bottom reef. This characteristic makes artificial breeding more difficult.

Method used

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  • Hexagrammos otakii spermatogonium culture medium and culture method
  • Hexagrammos otakii spermatogonium culture medium and culture method
  • Hexagrammos otakii spermatogonium culture medium and culture method

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Embodiment 1

[0023] Embodiment 1, the separation and purification of Otaki hexaline spermatogonia, the specific method is as follows:

[0024] Experimental reagents: triple antibody (BI), double antibody (BI), DMEM / F12 (BI), L-15 (Gibco), GDNF (Gibco), collagenase I (Sigma), collagenase IV (Sigma), Percoll ( Sigma), bFGF (Solarbio), fetal bovine serum (BI), L-glutamine (Thermo Fisher), sodium pyruvate (Sigma), bovine insulin (Solarbio), trypsin (Gibco), BCIP / NBT alkaline phosphate Enzyme Chromogenic Kit (Beyotime).

[0025] 1) Experiment preparation: Prepare disposable petri dishes, 15mL centrifuge tubes, scissors, tweezers, penicillin vials, pipette tips, etc., all of which must be sterile;

[0026] 2) Sampling and disinfection of the testicular tissue of Hexaline Otaki: ①Select a 13-month-old Hexaline Otaki to sample the testicular tissue. ② Before sampling, use 75% alcohol to disinfect the fish body, scissors, tweezers, etc., remove surface bacteria as much as possible, remove the tes...

Embodiment 2

[0029] The short-term in vitro culture of the spermatogonia of embodiment 2 Otaki six line fish

[0030] 1) Collect the cells obtained by Percoll and two differential attachment methods, place them on the supporting cells and culture them with the following medium with different components:

[0031] ①DMEM / F12+1%FBS+1% double antibody+1% fish serum+1% non-essential amino acid;

[0032] ②DMEM / F12+5%FBS+1% double antibody+1% fish serum+1% non-essential amino acid;

[0033] ③DMEM / F12+1%FBS+1% double antibody+1% fish serum+1% non-essential amino acid+10ng / mL GDNF+10ng / mL bFGF+10ng / mL bovine insulin+2mmol / L L-glutamine+1mmol / L sodium pyruvate;

[0034] ④DMEM / F12+5% FBS+1% double antibody+1% fish serum+1% non-essential amino acid+10ng / mL GDNF+10ng / mL bFGF+10ng / mL bovine insulin+2mmol / L L-glutamine+1mmol / L sodium pyruvate;

[0035] ⑤L-15+1%FBS+1% double antibody+1% fish serum+1% non-essential amino acid;

[0036] ⑥L-15+5% FBS+1% double antibody+1% fish serum+1% non-essential am...

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Abstract

The invention relates to a spermatogonial cell culture medium and a culture method of hexagrammos otakii, and belongs to the field of cell biology, the spermatogonial cell culture medium comprises the following components: a mixed culture medium, MEM non-essential amino acid with the volume ratio of 1%, double antibodies with the volume ratio of 1%, fish serum with the volume ratio of 1%, growth factors, nutrient substances and fetal calf serum with the volume ratio of 5%. The mixed culture medium is formed by mixing L-15 and DMEM / F12 according to the mass ratio of 1: 1. The culture medium and the culture method are used for culturing the Hexagrammos otakii spermatogonium which is separated and purified in vitro, the spermatogonium which is relatively high in purity and has dryness and proliferative activity can be obtained, and a basic experimental material is provided for research on a regulation mechanism of marine fish spermatogonium transplantation and spermatogonium proliferation and differentiation.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a spermatogonia culture medium and a culture method for in vitro short-term culture of the spermatogonia of Otaki hexaline fish. Background technique [0002] Spermatogonial stem cells (SSCs) are a type of adult stem cells with both proliferative and developmental potential. cells, which in males develop into male germ cells. However, the number of spermatogonial stem cells in the testis is scarce. Taking adult mice as an example, the number of spermatogonial stem cells only accounts for 0.02% to 0.03% of the total germ cells in the testis. Therefore, it is very important to establish an efficient method for the purification and cultivation of spermatogonia . [0003] Hexagrammos otakii belongs to the order Scorpius, Hexagrammosidae, and the genus Hexagrammos. It is a cold-temperate offshore bottom fish and a common economic species in the Yellow Sea, Bohai Sea and East ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2509/00C12N2509/10C12N2501/115C12N2501/13C12N2501/2315C12N2501/33C12N2500/30C12N2500/32
Inventor 齐洁贺艳赵茜
Owner OCEAN UNIV OF CHINA
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