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Culture method for in-vitro human oocyte preantral follicles

A technology of human oocytes and culture methods, which is applied to germ cells, animal cells, vertebrate cells, etc., and can solve problems such as difficult cultivation, low success rate, and instability

Inactive Publication Date: 2013-06-12
孔凡锋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the lack of proliferation and differentiation of granulosa cells in conventional preantral follicle culture leads to defects in follicle development. expected effects that affect further research on

Method used

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  • Culture method for in-vitro human oocyte preantral follicles
  • Culture method for in-vitro human oocyte preantral follicles
  • Culture method for in-vitro human oocyte preantral follicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] An in vitro human oocyte preantral follicle culture method, characterized in that the specific steps of the culture method include:

[0011] 1) Thaw the in vitro human oocytes stored in cryoprotective solution containing sucrose and propylene glycol and then resuscitate and culture them;

[0012] 2) The in vitro human oocytes obtained by the resuscitation culture are separated and extracted, and then placed in a nutrient solution containing follicular estrogen FSH for preantral culture.

[0013] Preferably, a certain amount of Trichostatin A (TSA) is added during the cultivation process.

[0014] Preferably, the specific method of step 2) is: configure the nutrient solution, add penicillin 110IU / ml, streptomycin 60μg / ml to the basic nutrient solution Ham'sF-10, and add 17% umbilical cord serum FCS, and add the concentration FSH is 2.3IU / ml follicular estrogen; the obtained in vitro human oocytes are washed with Ham'sF-10 containing 17% FCS and transferred to nutrient solution d...

Embodiment 2

[0019] The storage method is the same as in Example 1. The in vitro human oocytes obtained from the resuscitation culture were separated and extracted, and then placed in a nutrient solution containing follicular estrogen FSH for pre-antral culture. Configure the nutrient solution, add penicillin 100IU / ml, streptomycin 50μg / ml to the basic nutrient solution Ham'sF-10, add 15% umbilical cord serum FCS, and add follicular estrogen FSH with a concentration of 1IU / ml; A certain amount of Trichostatin A (TSA) is added during the culture process, and the concentration of Trichostatin A (TSA) in the nutrient solution is 100 μg / 1. The obtained in vitro human oocytes were washed with Ham'sF-10 containing 15% FCS and then transferred into droplets of nutrient solution, covered with mineral oil, in a carbon dioxide incubator at 37°C, 5% CO2, and saturated humidity Cultivate in medium and change the medium every 24 hours. The test results are as follows:

[0020] The effects of different ...

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Abstract

The invention provides a culture method for in-vitro human oocyte preantral follicles. The culture method is characterized by comprising the following specific steps of: 1) unfreezing the in-vitro human oocytes placed in frozen protecting solution containing cane sugar and propylene glycol and subjected to liquid nitrogen low-temperature storage, and then reviving and culturing; and 2) separating and extracting the in-vitro human oocytes obtained by reviving and culturing, then placing the in-vitro human oocytes in nutrient solution containing follicle-stimulating hormone (FSH), and performing preantral culture, wherein the culture for in-vitro human oocyte preantral follicles needs the support of FSH, and FSH can be used for promoting the growth of the follicles, increasing the survival time of the follicles, promoting the proliferation of granular cells, and promoting the maturation of the oocytes, as well as is an important factor for regulating and controlling the growth and development of the follicles; and trichostatin A can be used for effectively inhibiting the proliferation of tumour cells, regulating and controlling the progress of a cell cycle and then inducing the apoptosis of tumour cells, thus ensuring the survival quality of the cells. The method has important significance on researches in the aspect of test-tube babies.

Description

Technical field [0001] The invention belongs to the field of biomedicine technology and is an in vitro human oocyte preantral follicle culture method. Background technique [0002] In recent years, with the continuous development of embryo engineering technology, especially test tube baby technology, the demand for oocytes is increasing. The use of mature oocytes in the body can no longer meet the medical needs. People have begun to focus on the study of huge eggs. The development and utilization of preantral follicles with the potential of mother cell source. However, the lack of granulosa cell proliferation and differentiation during conventional preantral follicle culture leads to defects in follicular development. The survival time of follicles in the nutrient solution is very short, the cultivation is difficult, the success rate is low and unstable, making the preantral follicle culture unreachable The expected effect affects its further research. Summary of the invention ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
Inventor 孔凡锋
Owner 孔凡锋
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