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Culture method for in-vitro human oocyte preantral follicles

A technology of human oocytes and preantral follicles, which is applied in the field of in vitro human oocyte preantral follicle culture, which can solve the problems of difficult culture, short survival days of follicles, and low success rate

Inactive Publication Date: 2015-01-21
孔凡锋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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] A method for culturing preantral follicles of human oocytes in vitro, characterized in that the specific steps of the culturing method include:

[0011] 1) thawing and culturing in vitro human oocytes placed in a cryoprotectant solution containing sucrose and propylene glycol and stored in liquid nitrogen at a low temperature;

[0012] 2) The in vitro human oocytes obtained from resuscitation culture were separated and extracted, and then put into the nutrient solution containing follicular estrogen FSH for precavitary culture.

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

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

Embodiment 2

[0019] The storage method is the same as in Example 1. The in vitro human oocytes obtained from resuscitated culture were separated and extracted, and then put into the nutrient solution containing follicle estrogen FSH for precavitary culture. Configure nutrient solution, add penicillin 100IU / ml, streptomycin 50μg / ml in basic nutrient solution Ham'sF-10, add 15% umbilical cord serum FCS, and add follicle estrogen FSH with a concentration of 1IU / ml; A certain amount of Trichostatin A (TSA) was added during the cultivation process, and the concentration of Trichostatin A (TSA) in the nutrient solution was 100 μg / 1. The obtained in vitro human oocytes were washed in Ham'sF-10 containing 15% FCS and then transferred into microdroplets of nutrient solution. cultured and the medium was changed every 24 hours. The test results are as follows:

[0020] The effects of different concentrations of FSH on the in vitro growth days of human preantral follicles are shown in Table 1:

[...

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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 technical field of biomedicine and relates to a method for culturing preantral follicles of human oocytes in vitro. Background technique [0002] In recent years, with the continuous development of embryo engineering technology, especially the test-tube baby technology, the demand for oocytes is increasing day by day, and the use of oocytes matured in vivo can no longer meet medical needs. Development and utilization of preantral follicles of blastogenic potential. However, the lack of proliferation and differentiation of granulosa cells in conventional preantral follicle culture leads to defects in follicle development. The expected effect will affect its further research. Contents of the invention [0003] The object of the present invention is achieved like this: a kind of in vitro human oocyte preantral follicle culture method, its specific steps comprise: 1) will be placed in the cryoprotectant solution that contai...

Claims

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Application Information

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