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In-vitro cultivating maturation process of immaturation ovocyte through promoting discharging separating or obtained by sucking in ovarian tissue

A technology for in vitro culture of oocytes, applied in the field of cell culture, can solve the problems that limit the use and application of the advantages of IVM technology, the maturity rate of 90% of oocytes is not reached, and the nucleoplasmic maturation of oocytes is inconsistent, and the results are achieved. Stable, less traumatic, and easy-to-operate effects

Inactive Publication Date: 2011-08-31
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 maturation process of immaturation ovocyte through promoting discharging separating or obtained by sucking in ovarian tissue
  • In-vitro cultivating maturation process of immaturation ovocyte through promoting discharging separating or obtained by sucking in ovarian tissue
  • In-vitro cultivating maturation process of immaturation ovocyte through promoting discharging separating or obtained by sucking in ovarian tissue

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[0027] The culture medium of primary and secondary MSCs in the present invention is DMEM, and 10% fetal bovine serum, 10mmol / L HEPES, 100U / mL penicillin and 100U / mL streptomycin are added therein. Other mediums suitable for MSC growth, such as RPMI-1640, Ham'sF-12, etc., can also be used in the present invention.

[0028] The inventive method operates as follows:

[0029] 1. Isolation and culture of bone marrow mesenchymal stem cells MSCs from female mice: Take out the bilateral femurs and tibiae of 8-10 week old female Kunming mice under sterile conditions, cut off both ends of the bone, draw a heparinized syringe without serum The bone marrow cavity was repeatedly flushed with DMEM. Collect the rinsing solution, add it to the top of the sterile Percol solution with a density of 1.077 in the centrifuge tube in equal proportions, centrifuge at 2000 rpm for 20 minutes, collect the mononuclear cell layer between the two liquid layers, and centrifuge at 10 times the volume of 1×...

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Abstract

The method of obtaining immature oocyte from ovary tissue through separating or extracting for extracorporeal culture includes selecting marrow mesenchyme stem cell MSC of female mouse as the feeder cell or culturing the immature oocyte in the MSC culture liquid. After culturing, the co-culture system has obviously increased estradiol, progestin and luteotopin contents, and over 90 % of the immature oocyte become mature. 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 relates to a cell culture method, more specifically a method for in vitro culture and maturation of animal immature oocytes separated by stimulation or obtained by suction from ovarian tissue. 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. [0003] Culture medium is a key content of oocyte IVM technology, but there is no cheap, stable and safe commercial IVM culture medium available. At prese...

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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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