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Separation, primary culture and subculture method for granular cells in follicles in porcine ovary GV period

A technology of primary culture and subculture, applied in the field of cell culture, can solve the problems of non-adherent cells and turbid cells, and achieve the effects of high consistency of results, high cell uniformity and high feasibility

Pending Publication Date: 2021-05-28
DALI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] For this reason, the present invention provides a method for the separation, primary culture and subculture of follicular granulosa cells in the GV stage of porcine ovary, so as to solve the problem of cell turbidity and non-adhesion of cells in the prior art after the primary culture of porcine ovary granulosa cells. And other issues

Method used

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  • Separation, primary culture and subculture method for granular cells in follicles in porcine ovary GV period
  • Separation, primary culture and subculture method for granular cells in follicles in porcine ovary GV period
  • Separation, primary culture and subculture method for granular cells in follicles in porcine ovary GV period

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Embodiment

[0038] Embodiment A kind of method for separation, primary culture and subculture of granulosa cells in porcine ovary GV stage follicles

[0039] DMEM / F12 medium, WISENT INC., lot number: 319085029;

[0040] 0.25% trypsin (0.1% EDTA), WISENT INC, batch number: 325043027;

[0041] Penicillin-Streptomycin for cells, Penicillin-Streptomycin Solution, HyClone INC, batch number: J190007;

[0042] Fetal bovine serum FBS (Fetal Bovine Serum): gibco, batch number: 2166446;

[0043] PBS buffer, WISENT INC., batch number: 311010050;

[0044] Microscope: Olympus CX31, photo 10*10, objective lens and eyepiece are 10 times.

[0045] After the pigs are slaughtered in Dali, the pigs need to be burned on fire. The ovaries of the pigs (fired twice) used to eat raw hides are not taken, because the high temperature will reduce the activity of cells in the ovaries. The invention adopts pigs that are normally slaughtered and burned once to extract ovaries.

[0046] 1. Preparation before exper...

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Abstract

The invention discloses a separation, primary culture and subculture method for granulosa cells in follicles in a pig ovary GV period. The method comprises the following steps: collecting a cyst-free healthy pig ovary, and cleaning the ovary; separating granular cells; carrying out primary culture on the granular cells for 48 hours; changing liquid in a culture bottle for primary culture; after changing the liquid, continuously culturing the cells for 36 hours, and cleaning, digesting and terminating the cultured cells; and finally, sub-packaging and subculturing the cells for 48 hours. The invention provides a pollution-free ovary collection method, optimizes a granulosa cell acquisition and culture method, and further provides a primary granulosa cell subculture method. Granular cells obtained by the method are almost free of pollution, high in cell uniformity, high in consistency in the growth and development period, high in cell survival rate, high in success rate of making oxidative stress models and the like, and high in result consistency, practicability, stability and feasibility. In addition, the number of obtained cells is large, many subsequent experiments can be carried out, the number of times of going to a slaughter house is reduced, and the efficiency is high.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for the separation, primary culture and subculture of granulosa cells in follicles in the GV stage of porcine ovary. Background technique [0002] Granulosa cells can provide nutrients to oocytes, transmit signals, promote the maturation of oocytes, and improve the quality of eggs. Follicular atresia is a normal physiological phenomenon during follicular development. However, abnormal follicular atresia will reduce the number of available follicles, reduce the number of ovulations, and then reduce human reproductive function and pig litter size. Granulosa cell apoptosis is the main cause of follicular atresia, and oxidative stress can induce the apoptosis of porcine ovary granulosa cells. However, at present, there is no porcine ovary granulosa cell line on the market, and there is no normal human ovary granulosa cell line, and pig is a more suitable experimental animal mode...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2509/00
Inventor 隋世燕孔彩华王钦刘克娜龚绍荣
Owner DALI UNIV
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