CN112852712A - Method for separating, primary culturing and subculturing granulosa cells in pig ovary GV stage follicle - Google Patents
Method for separating, primary culturing and subculturing granulosa cells in pig ovary GV stage follicle Download PDFInfo
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Abstract
The invention discloses a method for separating granulosa cells in follicles of pig ovaries in GV stage, primary culture and subculture, which comprises the steps of collecting healthy ovaries of pigs without cysts, and cleaning the ovaries; isolating the granulosa cells; then carrying out primary culture on the granular cells for 48 h; replacing the culture flask for primary culture; continuously culturing for 36h after liquid replacement, and cleaning, digesting and stopping the cultured cells; finally, subpackaging and subculturing for 48 h. The invention provides a pollution-free ovary collection method, optimizes the granular cell obtaining and culturing method and also provides a primary granular cell subculture method. The granular cells obtained by the method disclosed by the invention are almost pollution-free, high in cell uniformity, high in consistency of growth and development periods, high in cell survival rate, high in success rate of making models of oxidative stress and the like, high in result consistency, strong in practicability, strong in stability and high in feasibility. In addition, the number of the obtained cells is large, subsequent experiments can be carried out more, the frequency of going to a slaughterhouse is reduced, and the efficiency is high.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a method for separating granulosa cells in follicles in the GV stage of pig ovaries, primary culture and subculture.
Background
The granular cells can provide nutrients for the oocytes, transmit signals, promote the maturation of the oocytes and improve the quality of the ova. Follicular atresia is a normal physiological event during follicular development, however, abnormal follicular atresia will reduce the number of available follicles, reduce the number of ovulations, and in turn reduce human reproductive function and reduce litter size in pigs. Granulosa cell apoptosis is the major cause of follicular atresia, and oxidative stress can induce apoptosis of pig ovarian granulosa cells. However, at present, no pig ovarian granulosa cell line exists in the market, and no normal human ovarian granulosa cell line exists, and pigs are suitable experimental animal models for human beings. Therefore, in vivo collection of pig ovarian granulosa cells for culture is an important technical basis for researching ovarian function.
However, in the prior art, the primary culture after the separation of the swine ovary granulosa cells has the phenomena of cell turbidity, cell non-adherence and the like, and the subculture cannot be performed for subsequent experiments.
Disclosure of Invention
Therefore, the invention provides a method for separating, primary culturing and subculturing granulosa cells in follicles in the GV stage of pig ovaries, and aims to solve the problems that the primary culturing is turbid in cells, cells are not attached to walls and the like after the separation of the granulosa cells of the pig ovaries in the prior art.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to the invention, the method for isolating, primarily culturing and subculturing the granulosa cells in the ovarian follicles in the GV stage of the pig ovaries comprises the following steps:
step one, collecting ovaries
Collecting ovaries of healthy pigs without cysts, and cleaning the ovaries;
step two, obtaining granular cell sediment
Taking 8-9mL of the ovarian cell fluid in a 15mL centrifuge tube, centrifuging for 5min at 1000r, and removing supernatant; adding 2ml PBS buffer solution, blowing, beating, mixing, centrifuging at 1000r for 5min, and removing supernatant; adding 2ml PBS buffer solution again, blowing, beating and mixing uniformly, centrifuging for 5min at 1000r, and removing supernatant; finally, 4mL of all-contained culture medium is added, blown, beaten and uniformly mixed to obtain granular cell suspension;
step three, primary culture
After 10. mu.L of the granular cell suspension was diluted 100-fold, the cells were counted under a microscope at 5X 108Inoculating each bottle into a T25 culture bottle, adding 4mL of all-contained culture medium into the culture bottle, blowing, beating and uniformly mixing, and culturing in an incubator to obtain primary culture cells;
step four, primary culture medium replacement
After culturing for 48h, removing the culture solution, cleaning adherent cells by using 2mL PBS buffer solution, removing the cleaning solution, adding 3mL of all-contained culture medium, and culturing in an incubator;
step five, subculturing
Culturing for 36h in an incubator, adding 3ml PBS buffer solution to clean adherent cells for 1-2 times after the anchorage rate reaches 70% -80%, and removing the cleaning solution; adding 1-2mL of pancreatin, and digesting for 2min in an incubator at constant temperature; adding a culture medium which is equal to the pancreatin in quantity to terminate the pancreatin reaction, and blowing, beating and uniformly mixing; transferring to a 15mL centrifuge tube, centrifuging for 5min at 1000r, removing supernatant, adding 1mL all-in-one culture medium, blowing, beating, mixing uniformly, and counting cells under a microscope; at 106Inoculating each bottle into a T25 culture bottle, adding 3mL of all-contained culture medium, and culturing in an incubator; obtaining subculture cells.
Further, in the first step, the ovary is washed with 0.9% physiological saline for 2 times, and washed with 75% alcohol for 1 time; washing with 0.9% physiological saline for 1 time, and washing with 75% alcohol for 2 times; and finally, washing the blood vessel with 0.9% of physiological saline for 2-3 times until no blood water exists.
Further, the total culture medium comprises 84mL of DMEM/F12 culture solution, 15mL of fetal bovine serum and 1mL of streptomycin for cells.
Further, the conditions of the incubator are 37 ℃ and 5% CO2。
Furthermore, in the second step, the method for taking the cell sap of the ovary is to use a 5mL disposable syringe with an upward needle hole to suck the cell sap of the ovary while pricking, so that the inner cell sap is not allowed to flow out.
Furthermore, the particle cell suspension is diluted 100 times by taking 10 μ L of the particle cell sediment into a 1.5mL centrifuge tube containing 990 μ L of PBS buffer solution, and then the mixture is blown and beaten uniformly.
Further, in the third step, the uniformly mixing is performed by lightly shaking the table top by adopting an 8-character method.
Further, the GV stage follicle means a follicle in which granulosa cells having a diameter of 3 to 5mm have not fully differentiated and matured.
Further, the PBS buffer is a PBS buffer formed by mixing 99ml PBS buffer and 1ml SK buffer.
The invention has the following advantages:
the invention provides a pollution-free ovary collection method, optimizes the granular cell obtaining and culturing method and also provides a primary granular cell subculture method. The granular cells obtained by the method disclosed by the invention are almost pollution-free, high in cell uniformity, high in consistency of growth and development periods, high in cell survival rate, high in success rate of making models of oxidative stress and the like, high in result consistency, strong in practicability, strong in stability and high in feasibility. In addition, the number of the obtained cells is large, subsequent experiments can be carried out more, the frequency of going to a slaughterhouse is reduced, and the efficiency is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
FIG. 1 is a diagram of a healthy pig ovary without cysts according to the present invention;
FIG. 2 is a diagram illustrating a method of removing follicles from an ovarian cavity according to the present invention;
FIG. 3 is a diagram of a healthy follicle provided by the present invention;
FIG. 4 is a diagram showing the state of cells adherent to 24h under a microscope according to the present invention;
FIG. 5 is a diagram showing the state of cells adherent for 48h under a microscope according to the present invention;
FIG. 6 is a diagram showing the cell anchorage state under a microscope after a primary culture medium is changed and cultured for 24 hours according to the present invention;
FIG. 7 is a microscopic image of a digested and inoculated T25 flask and mixed cells in subculture according to the present invention;
FIG. 8 is a microscopic cell image of a subculture cell of the present invention for 48 h.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example a method for isolating, primary culturing and subculturing granulosa cells in ovarian follicles in GV stage of pig ovary
DMEM/F12 medium, WISENT inc., lot No.: 319085029, respectively;
0.25% pancreatin (0.1% EDTA), WISENT INC, batch No.: 325043027, respectively;
cells were purified with Streptomycin, Penicillin-Streptomycin Solution, HyClone INC, lot number: j190007;
fetal Bovine serum fbs (total Bovine serum): gibco, lot number: 2166446, respectively;
PBS buffer, WISENT inc, lot number: 311010050, respectively;
microscope: olympus CX31, photograph 10 × 10, with 10 x objective and eyepiece.
After being slaughtered by a big pig, the pig needs to be burnt by fire, the ovary of the pig which eats the raw skin (twice burning) is not taken, and the activity of cells in the ovary is reduced due to the overhigh temperature. The invention adopts the pig which is normally slaughtered and then is burned once to take the ovary.
First, preparation before experiment
1. High-pressure sterilization experimental article
Gun heads (1 box 10. mu.L, 4 box 1mL, 1 box 200. mu.L, 1 box 5mL), 2 big beakers (1000mL), 5 250mL glass bottles, 15mL centrifuge tubes (unscrewing the cover, 20 pieces), 50mL centrifuge tubes (unscrewing the cover, 8 pieces), 1.5mL centrifuge tubes, 3 small scissors, 1 long tweezers and distilled water, and autoclaving at 121 ℃ and 147KPa for 30 min;
2. putting other articles except distilled water in an oven, and drying for 6h at 70 ℃;
3. solution preparation:
preparing a culture medium: 84mL of DMEM/F12 culture solution, 15mL of Fetal Bovine Serum (FBS) and 1mL of streptomycin for cells (SK) are uniformly mixed;
preparation of 1% SK-containing PBS buffer: 99ml PBS buffer and 1ml SK mixed well. The PBS buffer mentioned in the present invention is PBS buffer containing 1% SK.
4. The cell chamber was UV-sterilized for 30min in advance.
Secondly, separating granular cells
1) Preparing 1.5L of a vacuum flask, adding 1L of 0.9% normal saline, heating to 40 ℃, adding 160 ten thousand units of injection penicillin sodium and 100 ten thousand units of injection streptomycin sulfate, covering a cover, and turning upside down and mixing uniformly;
2) when the entrails of the butcher are removed, the person collecting the ovaries returns to the end of the rectum, the uterus and the ovaries cannot fall on the ground, the person collecting the ovaries is placed above the intestines, healthy ovaries without cysts are collected, and as shown in figure 1, the affiliated tissues are removed after collection and the ovaries are placed in the vacuum bottle;
3) opening the vacuum flask, pouring out part of the blood, and pouring the rest blood and the ovary into a big beaker A; cutting off ovarian collateral tissues, and putting into another big beaker B filled with 0.9% of normal saline;
4) washing the ovary in the big beaker B with 0.9% of normal saline for 2 times, washing with 75% of alcohol for 1 time, then washing with 0.9% of normal saline for 2 times, washing with 75% of alcohol for 1 time, finally washing with 0.9% of normal saline for 2-3 times until no blood exists, and putting the big beaker B into a water bath kettle containing 0.9% of normal saline;
5) taking granular cells: taking out an ovary from the big beaker B, and sucking cell sap while pricking with a 5mL disposable syringe with a needle hole upward to prevent the inner cell sap from flowing out as much as possible, as shown in figure 2; wherein the follicle selection standard is 3-5mm pale yellow transparent follicles, as shown in figure 3. When 1-2mL of cell sap exists in the injector, transferring the cell sap into a 15mL centrifuge tube, and repeating the steps until all the cell sap is taken out, wherein all the cell sap is taken out within 1 hour as far as possible;
6) centrifuging the centrifuge tube containing 8-9mL of cell sap at 1000r for 5 min; removing supernatant, adding 2ml PBS buffer solution, blowing, beating, mixing, centrifuging at 1000r for 5min, and removing supernatant; adding 2ml PBS buffer solution again, blowing, beating and mixing uniformly, centrifuging for 5min at 1000r, and removing supernatant; obtaining granular cell sediment;
7) adding 4mL of all-culture medium into the granular cell sediment, blowing, beating and uniformly mixing to obtain cell suspension; taking 10 mu L of cell suspension, and uniformly mixing the cell suspension into a 1.5mL centrifuge tube containing 990 mu L of PBS buffer solution, namely diluting the cell suspension by 100 times and counting under a microscope; at about 5X 108Inoculating each bottle into a T25 culture bottle, adding 4mL of all-contained culture medium into the culture bottle, covering the cover of the culture bottle (if the cover is not provided with holes, the cover is loosened by half circle), marking the name and time, and lightly shaking the table top to mix uniformly by using an 8-shaped method; at 37 deg.C, 5% CO2The incubator of (1); observing the cell attaching state at different times under a microscope, wherein the cell is attached for 24h, as shown in figure 4; cell state of cell adherence for 48hAs shown in fig. 5.
Thirdly, primary culture and subculture of granulosa cells
1. Primary culture
Culturing at 37 deg.C in 5% CO2 incubator for 48 hr, removing culture solution, washing adherent cells with 2mL PBS buffer solution, removing washing solution, adding 3mL total culture medium, incubating at 37 deg.C in 5% CO2 incubator2Continuing culturing; after 24h of culture, round, transparent and scattered granular cells can be seen under a microscope, as shown in FIG. 6;
2. subculturing
After culturing for 36h in an incubator, observing whether cells are polluted (turbid in a bottle, no cell form under a microscope, no or few adherent cells on the bottle wall), if no, removing the culture solution, adding 3mL PBS buffer solution to clean the adherent cells for 1-2 times, removing the cleaning solution, adding 2mL pancreatin, and culturing at 37 ℃ and 5% CO for 5% while ensuring that the cell adherence rate reaches 70% -80%, and then2Digesting in incubator for 2 min; taking out, adding a culture medium with the same amount as pancreatin to terminate pancreatin reaction, and blowing, beating and uniformly mixing; transferring the liquid to a 15mL centrifuge tube, centrifuging for 5min, removing supernatant, adding 1mL all-in-one culture medium, blowing, beating, mixing uniformly, and counting cells under a microscope; at about 106Inoculating each bottle into T25 culture flask, adding 3mL total culture medium, covering with cover (or half-circle if the cover is not porous), shaking gently on table top by "8" method, mixing, observing under microscope, and placing into incubator at 37 deg.C with 5% CO2And continuously culturing to obtain the subcultured cells. After 24-48h of culture, the color in the culture bottle is normal, a turbid state does not appear, subculture cells observed under a microscope are shown in figure 8, and the subsequent experiment can be carried out when the cell fusion rate reaches about 90%.
Counting: cover the counting plate with a cover slip, aspirate 10 μ L of cell suspension diluted 100 fold and add to the counting chamber from the side. Count with a counter under the microscope. Counting principle: the upper side and the left side are counted.
Except the culture bottle, 75% alcohol is sprayed before other things enter the biological safety cabinet; when any cover is opened in the cabinet, the flame of the alcohol lamp is passed; the biological safety cabinet is sprayed with 75% alcohol before and after use to be wiped for disinfection. The culture bottle is subjected to ultraviolet sterilization in the bag in advance, when the culture bottle is used, the nozzle of the culture bottle needs to be subjected to flame of the alcohol lamp, the cover is subjected to flame of the alcohol lamp, and the cover is required to be subjected to 1s of attention, so that the damage of a filter membrane on the cover is prevented. In the whole process, the reagent and the cells are kept at a certain distance from the flame of the alcohol lamp, so that the reagent and the cells are prevented from being damaged due to overhigh temperature.
The method for separating, primary culturing and subculturing the granulosa cells in the follicles in the GV stage of the pig ovary disclosed by the invention can be used for separating the granulosa cells in the follicles in the GV stage of the pig ovary and carrying out primary culturing, and can also be used for carrying out subculturing under the condition of a laboratory.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A method for isolating granulosa cells in follicles in the GV stage of pig ovaries, primary culture and subculture is characterized by comprising the following steps:
step one, collecting ovaries
Collecting ovaries of healthy pigs without cysts, and cleaning the ovaries;
step two, obtaining granular cell sediment
Taking 8-9mL of the ovarian cell fluid in a 15mL centrifuge tube, centrifuging for 5min at 1000r, and removing supernatant; adding 2ml PBS buffer solution, blowing, beating, mixing, centrifuging at 1000r for 5min, and removing supernatant; adding 2ml PBS buffer solution again, blowing, beating and mixing uniformly, centrifuging for 5min at 1000r, and removing supernatant; finally, 4mL of all-contained culture medium is added, blown, beaten and uniformly mixed to obtain granular cell suspension;
step three, primary culture
After 10. mu.L of the granular cell suspension was diluted 100-fold, the cells were counted under a microscope at 5X 108Inoculating each bottle into a T25 culture bottle, adding 4mL of all-contained culture medium into the culture bottle, blowing, beating and uniformly mixing, and culturing in an incubator to obtain primary culture cells;
step four, primary culture medium replacement
After culturing for 48h, removing the culture solution, cleaning adherent cells by using 2mL PBS buffer solution, removing the cleaning solution, adding 3mL of all-contained culture medium, and culturing in an incubator;
step five, subculturing
Culturing for 36h in an incubator, adding 3ml PBS buffer solution to clean adherent cells for 1-2 times after the anchorage rate reaches 70% -80%, and removing the cleaning solution; adding 1-2mL of pancreatin, and digesting for 2min in an incubator at constant temperature; adding a culture medium which is equal to the pancreatin in quantity to terminate the pancreatin reaction, and blowing, beating and uniformly mixing; transferring to a 15mL centrifuge tube, centrifuging for 5min at 1000r, removing supernatant, adding 1mL all-in-one culture medium, blowing, beating, mixing uniformly, and counting cells under a microscope; at 106Inoculating each bottle into a T25 culture bottle, adding 3mL of all-contained culture medium, and culturing in an incubator; obtaining subculture cells.
2. The method for isolating granulosa cells in ovarian follicles in GV stage of pig ovaries, primary culture and subculture according to claim 1, wherein in the first step, the ovaries are washed by washing the ovaries with 0.9% physiological saline for 2 times and 75% alcohol for 1 time; washing with 0.9% physiological saline for 2 times, and washing with 75% ethanol for 1 time; and finally, washing the blood vessel with 0.9% of physiological saline for 2-3 times until no blood water exists.
3. The method for isolating granulosa cells in ovarian follicles in the GV stage of pig ovaries, performing primary culture and subculture according to claim 1, wherein the total culture medium comprises 84mL of DMEM/F12 culture solution, 15mL of fetal bovine serum and 1mL of streptomycin for cells.
4. The isolated and original granulosa cells in pig ovary in GV stage according to claim 1The generation culture and subculture method is characterized in that the conditions of the incubator are 37 ℃ and 5% CO2。
5. The method for isolation, primary culture and subculture of granulosa cells in ovarian follicles at the GV stage of pig ovaries according to claim 1, wherein in the second step, the cell sap of the ovaries is taken out by using a 5mL disposable syringe, with a needle hole facing upward, while sucking the cell sap of the ovaries, without allowing the internal cell sap to flow out.
6. The method for isolating, primary culturing and subculturing the granulosa cells in the ovarian GV stage follicle of the pig according to claim 1, wherein the granulosa cell suspension is diluted by 100 times by taking 10 μ L of the granulosa cells, precipitating the granulosa cells into a 1.5mL centrifuge tube containing 990 μ L of PBS buffer solution, and pumping and mixing the cell suspension.
7. The method for isolation, primary culture and subculture of granulosa cells in ovarian follicles at GV stage of pig ovary according to claim 1, wherein in the third step, the uniform mixing is performed by using a 8-letter method and a gentle shaking mixing on a table top.
8. The method for isolation, primary culture and subculture of granulosa cells in the pig ovarian GV stage according to claim 1, wherein the GV stage follicle is a follicle in which granulosa cells having a diameter of 3 to 5mm have not been fully differentiated and matured.
9. The method for isolating granulosa cells in ovarian follicles at the GV stage of pig ovaries, performing primary culture and subculture according to claim 1, wherein the PBS buffer is a mixture of 99ml PBS buffer and 1ml SK buffer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113999811A (en) * | 2022-01-04 | 2022-02-01 | 北京市农林科学院 | Method for establishing granulosa cell in vitro model of follicular cyst |
| CN114015645A (en) * | 2021-11-23 | 2022-02-08 | 三养健康科技有限公司 | Method for culturing granular cells |
| CN114032207A (en) * | 2021-08-02 | 2022-02-11 | 四川农业大学 | Method for separating and culturing swine ovary granular cells |
| CN114438015A (en) * | 2022-01-28 | 2022-05-06 | 首都医科大学附属北京妇产医院 | Human granular cell line from ovary and preparation method and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1628857A (en) * | 2004-09-06 | 2005-06-22 | 刘玉来 | Follicular granulosa cell anti-senescence technology |
| JP2008228632A (en) * | 2007-03-19 | 2008-10-02 | Bayer Schering Pharma Ag | Somatic stem cell of living body having differentiation inducing capability to ovarian granulosa cell or ovarian theca cell |
| CN105886460A (en) * | 2016-04-21 | 2016-08-24 | 广东省农业科学院动物科学研究所 | Separation culture method of laying duck small yellow follicle granular cells |
| CN106701659A (en) * | 2016-10-31 | 2017-05-24 | 四川农业大学 | Porcine ovarian granulosa cell primary culture method |
| US20180251729A1 (en) * | 2015-09-17 | 2018-09-06 | Tokyo University Of Agriculture Educational Corporated | Culture method for differentiating primordial germ cells into functionally mature oocytes |
| CN108588005A (en) * | 2018-04-27 | 2018-09-28 | 四川农业大学 | A kind of isolated culture method of duck folliculus ovarii film layer cell |
| CN111040989A (en) * | 2020-01-19 | 2020-04-21 | 江苏农牧科技职业学院 | Separation culture method of duck ovarian granulosa cells |
| CN111760034A (en) * | 2020-06-29 | 2020-10-13 | 华南农业大学 | Application of DNMT1 gene in porcine ovarian granulosa cells |
-
2021
- 2021-02-08 CN CN202110173074.5A patent/CN112852712A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1628857A (en) * | 2004-09-06 | 2005-06-22 | 刘玉来 | Follicular granulosa cell anti-senescence technology |
| JP2008228632A (en) * | 2007-03-19 | 2008-10-02 | Bayer Schering Pharma Ag | Somatic stem cell of living body having differentiation inducing capability to ovarian granulosa cell or ovarian theca cell |
| US20180251729A1 (en) * | 2015-09-17 | 2018-09-06 | Tokyo University Of Agriculture Educational Corporated | Culture method for differentiating primordial germ cells into functionally mature oocytes |
| CN105886460A (en) * | 2016-04-21 | 2016-08-24 | 广东省农业科学院动物科学研究所 | Separation culture method of laying duck small yellow follicle granular cells |
| CN106701659A (en) * | 2016-10-31 | 2017-05-24 | 四川农业大学 | Porcine ovarian granulosa cell primary culture method |
| CN108588005A (en) * | 2018-04-27 | 2018-09-28 | 四川农业大学 | A kind of isolated culture method of duck folliculus ovarii film layer cell |
| CN111040989A (en) * | 2020-01-19 | 2020-04-21 | 江苏农牧科技职业学院 | Separation culture method of duck ovarian granulosa cells |
| CN111760034A (en) * | 2020-06-29 | 2020-10-13 | 华南农业大学 | Application of DNMT1 gene in porcine ovarian granulosa cells |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114032207A (en) * | 2021-08-02 | 2022-02-11 | 四川农业大学 | Method for separating and culturing swine ovary granular cells |
| CN114015645A (en) * | 2021-11-23 | 2022-02-08 | 三养健康科技有限公司 | Method for culturing granular cells |
| CN113999811A (en) * | 2022-01-04 | 2022-02-01 | 北京市农林科学院 | Method for establishing granulosa cell in vitro model of follicular cyst |
| CN113999811B (en) * | 2022-01-04 | 2022-04-05 | 北京市农林科学院 | Method for establishing granulosa cell in vitro model of follicular cyst |
| CN114438015A (en) * | 2022-01-28 | 2022-05-06 | 首都医科大学附属北京妇产医院 | Human granular cell line from ovary and preparation method and application thereof |
| CN114438015B (en) * | 2022-01-28 | 2022-11-22 | 首都医科大学附属北京妇产医院 | Human granular cell line from ovary and preparation method and application thereof |
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