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In-vitro cultivating matural process of immatural ovocyte in ovarium organized block

A technology of ovarian tissue and in vitro culture, applied in the fields of reproductive medicine and cell biology, can solve the problems of not reaching 90% oocyte maturation rate, limiting the advantages and application of IVM technology, and inconsistency of nucleoplasmic maturation of oocytes , to achieve stable results, easy operation, and maintain tissue activity

Inactive Publication Date: 2011-08-03
ANHUI PROVINCIAL HOSPITAL
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Problems solved by technology

But these substances are very expensive and unstable, prone to inconsistent nucleoplasmic maturation of oocytes
Cambridge England "Reproduction" magazine (Reproduction, 2001, 121:925) reported that monkey kidney cells, granulosa cells and oviduct epithelial cells were used as feeder cells, and the secretion of cells was used to reduce the addition of substances and improve the maturation rate of oocytes. The 90% maturation rate of oocytes during ovulation stimulation has not been reached, and there is also the risk of contamination by pathogens and foreign genes
[0004]The existence of the above reasons greatly limits the use and application of the advantages of IVM technology

Method used

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  • In-vitro cultivating matural process of immatural ovocyte in ovarium organized block
  • In-vitro cultivating matural process of immatural ovocyte in ovarium organized block
  • In-vitro cultivating matural process of immatural ovocyte in ovarium organized block

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Embodiment Construction

[0028] The inventive method operates as follows:

[0029] 1. Isolation and culture of MSC: Obtain the bone marrow cavity flushing solution of 8-10 weeks old female mice, add equal proportions to the top of the sterile Percol solution with a density of 1.077 in the centrifuge tube, centrifuge at 2000 rpm for 20 minutes, and collect the two liquid layers The mononuclear cell layer in between was washed twice by centrifugation at 1200 rpm for 10 minutes with 10 times the volume of 1×PBS. Cells resuspended in DMEM containing 10% fetal bovine serum by volume, 2×10 6 / ml cell density inoculate culture flasks at 37°C in 5% CO by volume 2 to cultivate. After 3 days, gently shake the culture flask, pour off the non-adherent cells, wash 2 times with 1×PBS to obtain the purified primary MSC, add DMEM containing 10% fetal bovine serum by volume and continue to culture until 80% of the bottom of the flask is covered ( figure 1 shown), the weight volume ratio was 0.25% trypsin digestion ...

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Abstract

The extracorporeal culture and mature method of immature oocyte from ovary tissue block includes selecting marrow mesenchyme stem cell MSC of female mouse as the feeder cell, compounding MSC culture liquid and agar and covering the culture rack, adding HCG in 3 IU / ml, and adhering the ovary tissue block on the rack to culture. In the co-culture system, estradiol, progestin and luteotopin contentsare increased obviously, and after culturing, there is mature oocyte discharged. The method is significant in surviving rear endangered species of animals, breeding fine variety of animal, cell biological research of embryo stem cell, etc.

Description

technical field [0001] The invention belongs to the fields of reproductive medicine and cell biology, and particularly relates to a method for culturing and maturing immature oocytes in ovarian tissue blocks in vitro. Background technique [0002] Immature oocyte in vitro maturation (IVM) technology emerged with the development of artificial assisted reproductive technology (ART). IVM can shorten the reproductive cycle, maximize the use of good genetic material, and be used for artificial reproduction of rare or endangered animals. and the cultivation and commercial production of livestock breeds; it is convenient to study the development mechanism of oocytes, combined with in vitro fertilization or parthenogenetic activation technology to obtain a large number of embryonic stem cells for scientific research; IVM broadens the source of oocytes in the field of human reproduction and immature eggs Mother cells are easier to cryopreserve and help create an "egg bank" that provi...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/075
Inventor 冯定庆凌斌高婷周颖沈国栋朱园园姚凤球陈峥峥
Owner ANHUI PROVINCIAL HOSPITAL
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