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A cryopreservation solution of sturgeon gonad tissue and a method for cryopreservation and recovery

A cryopreservation solution and cryopreservation technology, applied in the preservation of human or animal body, animal cells, animal husbandry, etc., can solve the problems of complex cryopreservation solution components and freezing procedures, unknown reproductive stem characteristics of testicular cells, etc. Achieve long-term effective preservation, high activity, and simple formula components

Active Publication Date: 2021-11-30
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study used cryopreserved cells as the carrier, and did not involve the study of gonad tissue; and the components of the cryopreservation solution and the freezing procedure were complicated; the most important key point: "Whether the recovered testicular cells have the characteristics of reproductive stem cells" also unknown

Method used

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  • A cryopreservation solution of sturgeon gonad tissue and a method for cryopreservation and recovery
  • A cryopreservation solution of sturgeon gonad tissue and a method for cryopreservation and recovery
  • A cryopreservation solution of sturgeon gonad tissue and a method for cryopreservation and recovery

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The research on the composition of embodiment 1 cryopreservation solution

[0036] The composition of the cryopreservation solution was screened as follows:

[0037] 1. Screening of osmotic protectants (dimethyl sulfoxide, ethylene glycol and methanol) in cryopreservation solution components of sturgeon gonad tissue, the specific steps are as follows:

[0038]1. Use L-15 medium to prepare 5 ml of dimethyl sulfoxide, ethylene glycol, and methanol at three concentrations (1.0mol / L, 1.3mol / L, and 1.6mol / L) to obtain cryopreservative solutions.

[0039] 2. Dissect the 2-year-old sturgeon, put the testis and ovary into different petri dishes on ice, and wash the blood clots with pre-cooled PBS.

[0040] 3. Add an appropriate amount of L-15 to culture the culture-based culture dish, remove the peritoneum and fat on the testis and ovary, weigh the testis and ovary, and take 3 g of the testis and ovary respectively; cut the testis and ovary with scissors Shredded to 1 mm 3 A...

Embodiment 2

[0064] Embodiment 2 Cryopreservation and resuscitation of gonad tissue of sturgeon

[0065] details as follows:

[0066] 1. According to the results of Example 2, the preparation of cryopreservation solution includes: L-15 medium, 1.3mol / L dimethyl sulfoxide, 0.1mol / L trehalose and 100 ml of 10% fetal bovine serum. Dissect the 2-year-old sturgeon, put the testis and ovary into different petri dishes on ice, and wash the blood clots with pre-cooled PBS.

[0067] 2. Add an appropriate amount of L-15 to culture the culture-based culture dish, remove the peritoneum and fat on the testis and ovary, weigh the testis and ovary, and take 3 g of the testis and ovary respectively; cut the testis and ovary with scissors Shredded to 1 mm 3 About the size, the tissue pieces were washed with about 3 times the volume of L-15 medium, centrifuged at 200 g at 4°C for 5 min, and repeated 3 times.

[0068] 3. Add 50 ml of cryopreservation solution to the testis and ovary, mix well and transfer...

Embodiment 3

[0073] Example 3 Cryopreservation and Recovery of Chinese Sturgeon Gonad Tissue

[0074] details as follows:

[0075] 1. Prepare cryopreservation solution containing 50 ml of L-15 medium, 1.3mol / L dimethyl sulfoxide, 0.1mol / L trehalose and 10% fetal bovine serum. Dissect four 3-year-old male Chinese sturgeons, put the testis into a petri dish, and wash the blood clot with pre-cooled PBS.

[0076] 2. Add an appropriate amount of L-15 to culture in a petri dish, remove the peritoneum and fat on the testis, weigh the testis, take 3g testis; use scissors to cut the testis to 1mm 3 About the size, the tissue block was washed with about 3 times the volume of L-15 medium, centrifuged at 200 g at 4°C for 5 min, and repeated 3 times.

[0077] 3. Add 50 ml cryopreservation solution to the testis, mix well and transfer to a cryopreservation tube. After placing the cryovials and the programmed cooling box in ice for 1 h, the cryopreserved tubes were transferred to the programmed coolin...

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Abstract

The invention provides a cryopreservation solution of sturgeon gonad tissue and a method for cryopreservation and recovery. The present invention first takes the cultured fish as the object, systematically researches and screens out a cryopreservation solution with relatively simple components and a simple cryopreservation method, and proves the reproductive stem cell characteristics of gonad cells prepared after resuscitation through germ cell transplantation experiments , and also applied this method to the cryopreservation of the testis of the endangered fish Acipenser sinensis. Utilize the method provided by the present invention to carry out ultra-low temperature cryopreservation of sturgeon gonad tissue, prepare gonad single cell suspension after recovery, the cell activity can reach more than 90%, and there is no difference in the activity of cells prepared from fresh gonads; further effective through germ cell transplantation technology The germline stem cell properties of gonad cells were assessed. The cryopreservation solution of the invention has simple components and convenient operation of the cryopreservation method, and provides a new and effective way for long-term preservation and species recovery of endangered sturgeon germplasm resources.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cryopreservation liquid of sturgeon gonad tissue and a method for cryopreservation and recovery. Background technique [0002] At present, there are 27 species in the world, all of which are listed in Appendix I or II of the Convention on International Trade in Endangered Animals and Plants. Their wild populations are all endangered to varying degrees, and some are even extinct. Although a breakthrough has been made in the artificial reproduction of many species of sturgeon, most of them are large in size (up to several hundred kilograms in weight), take a long time for the first sexual maturity (more than ten years), and have different maturation between male and female. As a result, a large amount of manpower, material resources and financial resources need to be invested in the breeding and seed conservation process, resulting in huge breeding costs and extremely li...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02C12N5/071
CPCA01N1/0221C12N5/0608
Inventor 叶欢李创举危起伟周从利岳华梅阮瑞杜浩武梦斌
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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