Separation and culture method of ovarian granular cells of northern Guizhou Ma goats

A Qianbei hemp sheep and granular cell technology, applied in the field of cell biology, can solve the problems of few cleaning times, no clear regulation of cleaning times, probability of cell contamination, etc., to improve accuracy, easy operation, and fewer dead cells. Effect

Inactive Publication Date: 2021-02-05

GUIZHOU UNIV

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Problems solved by technology

[0004] At present, in the process of separating ovarian granulosa cells, there is no clear regulation on the number of times o...

Abstract

The present invention discloses a separation and culture method of ovarian granular cells of northern Guizhou Ma goats. The separation and culture method of ovarian granular cells of northern GuizhouMa goats comprises the following steps: sacrificing adult multiparous purebred northern Guizhou Ma goats by neck bloodletting, taking out ovaries, flushing away bloodstain, bringing the treated ovaries back to a sterile room, performing soaking with alcohol, rinsing with alcohol, normal saline and sterilized PBS, puncturing follicles with a needle, releasing ovarian granular cells, performing rinsing with a 10% complete medium, after centrifuging, taking a precipitate, continuously rinsing the precipitate with a sterilized PBS buffer solution, adding a 10% complete medium, and peforming blowing, beating, uniformly mixing, and culturing in a constant-temperature incubator. The pure ovarian granular cells can be separated through the method and the method is easy and convenient to operate, stable, and reliable and has good repeatability.

Application Domain

Cell dissociation methodsArtificial cell constructs +1

Technology Topic

Granular cellBiomedical engineering +10

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Experimental program(5)
Comparison scheme(4)

Example Embodiment

[0039]Example 1

[0040]The Qianbei Ma sheep ovarian granulosa cell isolation and culture method includes the following steps:

[0041](1) The adult purebred Qianbei Ma sheep ewes were sacrificed by neck bloodletting, the ovaries and follicles were separated, 75% alcohol and sterilized under 110℃, 100kPa conditions for 35 minutes and added 1:1 After rinsing blood stains with the PBS buffer of the streptomycin mixed solution, rinse 3 times and transfer to a beaker containing PBS buffer pre-cooled to 4℃, and quickly transfer to a sterile room within 4 hours, and maintain the temperature during the transfer process At 4°C;

[0042](2) Take out the ovaries and follicles obtained in step (1), soak them in 75% alcohol for 30s, press 75% alcohol-physiological saline-sterilized PBS buffer (including 1:1 penicillin and streptomycin solution) -DME/F12 medium is washed sequentially 3 times;

[0043](3) Place the ovaries and follicles treated in step (2) in a petri dish containing 100ml of 10% complete medium. The medium is composed of Australian fetal bovine serum with a volume ratio of 10:90:1, DMEM/ F12 medium, a mixture of penicillin and streptomycin (the penicillin content in the penicillin and streptomycin mixture is 10ku/ml, the streptomycin content is 10mg/ml, and the solvent is 0.9% NaCl solution). The largest follicle is pierced with a sterile syringe needle to release the granular cells, and rinsed with 10% complete medium to obtain a rinse solution;

[0044](4) Put the rinsing solution obtained in step (3) in a centrifuge tube, centrifuge at 2000 rpm for 10 minutes, and discard the supernatant;

[0045](5) Rinse the remaining precipitate in step (4) with sterile PBS buffer containing double antibody solution for 3 times;

[0046](6) Add 10% complete medium to the precipitate after rinsing in step (5) and mix it by pipetting, transfer it to a cell culture flask, and place it at 35°C, CO2Culture in a constant temperature incubator with a concentration of 8%, and change the medium every 24h.

Example Embodiment

[0047]Example 2

[0048]The Qianbei Ma sheep ovarian granulosa cell isolation and culture method includes the following steps:

[0049](1) The adult purebred Qianbei Ma sheep ewes were sacrificed by neck bloodletting, the ovaries and follicles were separated, and 75% alcohol was used and sterilized under the conditions of 120°C and 100kPa for 20 minutes and added 1:1 green After rinsing blood stains with the PBS buffer of the streptomycin mixed solution, rinse 3 times and transfer to a beaker containing PBS buffer pre-cooled to 4℃, and quickly transfer to a sterile room within 5 hours. The temperature is maintained during the transfer process At 8°C;

[0050](2) Take out the ovaries and follicles obtained in step (1), soak them in 75% alcohol for 30s, press 75% alcohol-saline-sterilized PBS buffer (including 1:1 penicillin, streptomycin solution) -Sequential washing of DME/F12 medium 5 times;

[0051](3) Place the ovaries and follicles treated in step (2) in a petri dish containing 100ml of 10% complete medium. The medium is composed of Australian fetal bovine serum with a volume ratio of 10:90:1, DMEM/ F12 medium, a mixture of penicillin and streptomycin (the penicillin content in the penicillin and streptomycin mixture is 10ku/ml, the streptomycin content is 10mg/ml, and the solvent is 0.9% NaCl solution). The largest follicle is pierced with a sterile syringe needle to release the granular cells, and rinsed with 10% complete medium to obtain a rinse solution;

[0052](4) Put the washing solution obtained in step (3) in a centrifuge tube, centrifuge at 2000 rpm for 15 minutes, and discard the supernatant;

[0053](5) Rinse the remaining precipitate in step (4) with sterile PBS buffer containing double antibody solution for 4 times;

[0054](6) Add 10% complete medium to the precipitate after rinsing in step (5) and mix it by pipetting, transfer it to a cell culture flask, and place it at 37℃, CO2Culture in a constant temperature incubator with a concentration of 5%, and replace the medium every 24h.

Example Embodiment

[0055]Example 3

[0056]The Qianbei Ma sheep ovarian granulosa cell isolation and culture method includes the following steps:

[0057](1) The adult purebred Qianbei Ma sheep ewes were sacrificed by neck bloodletting, the ovaries and follicles were separated, and 75% alcohol was used in sequence and sterilized under the conditions of 115°C and 90kPa for 30 minutes and added 1:1 green After rinsing blood stains with the PBS buffer of the streptomycin mixed solution, rinse 3 times and transfer to a beaker containing PBS buffer pre-cooled to 4℃, and quickly transfer to a sterile room within 4.8h. Temperature during transfer Keep at 7℃;

[0058](2) Take out the ovaries and follicles obtained in step (1), soak them in 75% alcohol for 30s, press 75% alcohol-saline-sterilized PBS buffer (including 1:1 penicillin, streptomycin solution) -Sequential washing of DME/F12 medium 5 times;

[0059](3) Place the ovaries and follicles treated in step (2) in a petri dish containing 100ml of 10% complete medium. The medium is composed of Australian fetal bovine serum with a volume ratio of 10:90:1, DMEM/ F12 medium, a mixture of penicillin and streptomycin (the penicillin content in the penicillin and streptomycin mixture is 10ku/ml, the streptomycin content is 10mg/ml, and the solvent is 0.9% NaCl solution). The largest follicle is pierced with a sterile syringe needle to release the granular cells, and rinsed with 10% complete medium to obtain a rinse solution;

[0060](4) Put the rinsing solution obtained in step (3) in a centrifuge tube, centrifuge at 1800 rpm for 12 minutes, and discard the supernatant;

[0061](5) Rinse the remaining precipitate in step (4) with sterile PBS buffer containing double antibody solution for 4 times;

[0062](6) Add 10% complete medium to the precipitate after rinsing in step (5) and mix it by pipetting, transfer it to a cell culture flask, and place it at a temperature of 36℃, CO2Culture in a constant temperature incubator with a concentration of 7%, and replace the medium every 24h.

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Separation and culture method of ovarian granular cells of northern Guizhou Ma goats

A Qianbei hemp sheep and granular cell technology, applied in the field of cell biology, can solve the problems of few cleaning times, no clear regulation of cleaning times, probability of cell contamination, etc., to improve accuracy, easy operation, and fewer dead cells. Effect

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