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Porcine ovarian granulosa cell primary culture method

A technology of granulosa cells and ovaries, applied in cell dissociation methods, animal cells, tissue culture, etc., can solve the problems of impure cell survival rate and time-consuming, and achieve the goal of shortening in vitro operation time, improving survival rate and avoiding pollution Effect

Pending Publication Date: 2017-05-24
SICHUAN AGRI UNIV
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Problems solved by technology

There are different in vitro primary culture methods of porcine ovarian granulosa cells reported in the existing literature, but most of them have problems such as time-consuming, impure or low cell viability

Method used

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  • Porcine ovarian granulosa cell primary culture method
  • Porcine ovarian granulosa cell primary culture method
  • Porcine ovarian granulosa cell primary culture method

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Embodiment Construction

[0026] The present invention will be specifically introduced below in conjunction with the accompanying drawings and specific embodiments.

[0027] 1. Prepare the solution

[0028] Solution I: 1×PBS solution.

[0029] Solution II: DMEM / F12 culture solution containing 1×antibiotics.

[0030] Solution III: Cell complete growth medium.

[0031] The preparation method of the complete cell growth culture medium (Solution III): mix the DMEM / F12 culture medium and FBS according to the volume ratio of 9:1, then add 1× antibiotics, and mix evenly.

[0032] 2. Collection of ovaries

[0033] Collect ovaries from puberty gilts at a local slaughterhouse, place the ovaries in an insulated container filled with sterile solution I at 37°C, and send them to the laboratory within two hours.

[0034] 3. Extraction of follicular fluid

[0035] Use a 20ml, 18g needle syringe to insert a needle into the follicle at a distance of 0.5mm from the base of the follicle, aspirate the follicle fluid,...

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Abstract

The invention discloses a porcine ovarian granulosa cell primary culture method. The method comprises steps as follows: ovaries are collected, follicular fluids are sucked, and cells are centrifugally collected; the cells are re-suspended and centrifugally collected to be dispersed, and the process is repeated 2-3 times; the cells are placed in an incubator for culture; solution replacement is performed for the cells after 24 h of culture, a solution, anchorage-independent cells and oocytes are sucked, rinsing is performed once, a fresh solution is added for continuous culture, and the solution is replaced once after 24 h of culture of cells. The method has the benefits as follows: (1) in the granulosa cell collecting process, the step of picking of oocytes with a mouth pipette under a stereomicroscope is omitted, the oocytes suspend to grow in the culture solution, a few of oocytes in a culture dish are sucked away with solution replacement after 24 h of culture, and adherent granulosa cells are pure; (2) the in-vitro operation time is shortened, and the survival rate of the granulosa cells is increased; (3) the obtained granulosa cells is purer.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a method for primary culture of porcine ovary granulosa cells, and belongs to the technical field of cell separation, purification and culture. Background technique [0002] Ovarian granulosa cells are cells differentiated from theca cells on the inner wall of the follicular cavity with the expansion of the follicular cavity and the increase of follicular fluid during the development of the ovarian secondary follicle to the cystic follicle, because the cytoplasm of this cell contains Granule-like substance, so it is called granule cells. There is a two-way exchange of signals and substances between granulosa cells and oocytes, and granulosa cells support and nourish the growth and development of oocytes. [0003] Granulosa cells are an important model for studying the biology of female germ cells and their functional regulation, and pigs and humans are highly homologous in genetics. The ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2509/00
Inventor 徐盛玉王定越吴德林燕车炼强方正锋
Owner SICHUAN AGRI UNIV
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