Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof

A vitrification and protection solution technology, which is applied in the preservation, application, and animal husbandry of human or animal bodies, and can solve problems such as the method of vitrification and protection solution freezing for large ovarian cortex slices, and achieve good cryopreservation effect , shorten transplantation, improve the effect of follicle survival

Inactive Publication Date: 2012-11-14
NINGXIA MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no cryoprotective solution and freezing method suitable for the vitrification of large ovarian cortex slices

Method used

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  • Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof
  • Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof
  • Ovarian large cortex piece vitrified cryopreservation protection liquid and cryopreservation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Method for vitrification of large ovarian cortex slices

[0045] (1) Vitrification

[0046] The large cortex samples of sheep ovaries aged 2 to 6 months were fixed to a thickness of 2 mm, and were divided into:

[0047] Sample (40mm 3 =5mm×4mm×2mm),

[0048] Medium sample (80mm 3 =10mm×4mm×2mm),

[0049] Large sample (160mm 3 =10mm×8mm×2mm),

[0050] Two-step osmotic equilibrium method was used for vitrification, and the whole process was carried out at room temperature (22-24°C):

[0051] The first step is pre-osmosis balance: 1.5mol / L ethylene glycol (EG) + 20% calf serum (ICS), small sample: 15min, medium sample: 20min, large sample: 25min;

[0052] The second step is vitrification osmotic balance: EG 5.5 / 30 solution (the specific formula of EG 5.5 / 30 solution is: EG+30% polysucrose+0.5mol / L sucrose), small sample: 5min~8min, medium sample: 10min~13min , Large sample: 17min~20min (the vitrification penetration equilibrium time of each cortical slic...

Embodiment 2

[0065] Example 2: Vitrification of sheep ovary large cortex slices

[0066] In the present invention, suitable gonadotropins are added to vitrification solution of 5.5mol / L EG+30% polysucrose+0.5mol / L sucrose. Among them, sheep were supplemented with 10 μg / mL FSH. Compared with the group without gonadotropin, no matter in vitro culture or in vivo transplantation after freezing, the follicle development, endothelial growth factor increased, and the number of surviving follicles in the group with gonadotropin were significantly different.

[0067] 1.1 Materials

[0068] 1.1.1 Sheep ovary

[0069] Sheep ovaries came from discarded ovaries of 2-6 month-old sheep slaughtered in Najiahu Halal Beef and Mutton Slaughterhouse, Yongning County, Yinchuan City, Ningxia, and were sent back to the laboratory in sterile culture medium at 4°C within 2 hours after slaughter.

[0070] 1.1.2 Experimental animals

[0071] BALB / CA male nude mice aged 8-12 weeks were provided by Beijing Weitong...

Embodiment 3

[0102] Example 3: Relevant research on the reduction of follicle apoptosis by FSH intervention at different periods of vitrified cryopreservation of mouse ovaries

[0103] In the present invention, suitable gonadotropin is added to the vitrification solution of 5.5 mol / L EG+30% polysucrose+0.5 mol / L sucrose. The mice were supplemented with 0.3IU / mL FSH. As a survival factor of follicles, FSH not only has an anti-apoptotic effect, but also participates in the development and maturation of follicles. The development of follicles is inseparable from the cell communication between gap junctions, and Cx43 can enhance this intercellular signal exchange. Therefore, this study intervened with FSH in different stages of vitrification, and observed the protective effect of FSH on ovarian vitrification by detecting the expression of Cx43 and activated caspase-3 (active caspase-3).

[0104] 1.1 Materials

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Abstract

The invention relates to an ovarian large cortex piece vitrified cryopreservation protection liquid and a cryopreservation method thereof. Although the vitrified cryopreservation method is widely applied to experimental animals, a universal cryopreservation method and a standard cryopreservation liquid preparation scheme are absent at present, so that the sized transplantation subjected to vitrified cryopreservation is at a small-size stage, an ovarian small cortex piece has limited follicles, and the service life of the transplanted follicles is influenced. The invention provides vitrified cryopreservation protection liquid suitable for an ovarian large cortex piece, and gonadotropin is added on the basis of the common vitrified liquid. According to the vitrified cryopreservation protection liquid suitable for the ovarian large cortex piece, especially the ovaries of experimental animals, improved variety livestock and wild endangered species in sudden death are conveniently cryopreserved at the most proper osmotic equilibrium time of performing vitrified cryopreservation on the ovarian large cortex pieces of different sizes; and moreover, according to the method, the survival rate of the follicles can be improved, the blood reperfusion time of the transplant is shortened, and the survival rate of the cryopreserved ovarian transplantation in vivo is improved.

Description

technical field [0001] The invention relates to the technical field of cryopreservation of cells, tissues and organs, in particular to a vitrification cryoprotective solution and a freezing method for large ovarian cortex slices. Background technique [0002] Vitrification is a new technology of ultra-fast ultra-low temperature preservation first proposed by Rall and Fahy in 1985. It is not only easy to operate, but also has a good freezing effect. It can better preserve the biological activity of living cells, tissues and organs. The advantages of economy, simplicity and high efficiency are one of the fastest-growing research directions in the field of cryobiology in recent years. [0003] The ovaries of cancer patients are cryopreserved before cancer radiotherapy and chemotherapy. After the cancer is controlled, the cryopreserved ovaries are transplanted back into the body to restore the endocrine function and fertility of the ovaries of the patients to a certain extent. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 王燕蓉党玲王红燕
Owner NINGXIA MEDICAL UNIV
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