Method for separating and culturing ovarian granular cells

Abstract

The invention provides a method for separating and culturing ovarian granular cells, and belongs to the technical field of cell biology. The method for separating and culturing the ovarian granular cells comprises the following steps: selecting follicles of 3-8mm in ovarian tissues; collecting follicular fluid by a suction method, and filtering impurities; collecting filtrate, centrifuging and taking precipitate to obtain ovarian granular cells; and resuspending the ovarian granulosa cells by using a DMEM / F12 complete medium, culturing the cell suspension, and subculturing when the density of the ovarian granulosa cells reaches 80-90%. The method for separating and culturing the ovarian granulosa cells is high in operability and simple, the extracted ovarian granulosa cells are few in parenchyma cells, the growth state is good after passage, the physiological state is stable, and the passage frequency is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and particularly relates to a method for isolating and culturing ovarian granulosa cells. Background Art

[0002] The orderly development of dairy cow follicles is closely related to the maintenance of female reproductive lifespan. The normal development of follicles is a prerequisite for reproductive activities such as estrus and ovulation in dairy cows. Therefore, extending the reproductive lifespan of dairy cows has become a technical bottleneck that urgently needs to be broken through for the healthy development of the dairy farming industry. As an important somatic cell component during follicle development, ovarian granulosa cells (GCs) maintain the physiological functions of the ovary by secreting gonadal hormones, namely progesterone and estrogen, and play a key regulatory role in the processes from primordial follicle growth initiation, oocyte maturation, steroid hormone synthesis, follicular atresia / ovulation to corpus luteum formation. Therefore, more and more evidence shows that the normal development of granulosa cells is the basis for ensuring ovarian and follicular development, is closely related to the reproductive performance of dairy cows, and occupies a very important position in various reproductive technologies. If granulosa cells have functional disorders, it will cause abnormal follicle development, and then lead to infertility. Therefore, in mammals, follicular granulosa cells are often used as a model for in vitro study of ovarian function.

[0003] As is well known, primary cells retain the characteristics and functions of in vivo tissues, can better reflect the physiological state in the organism, and provide a model closer to the physiological environment. However, the extraction process of primary cells is complex, it is difficult to obtain high-quality cell samples, the growth rate of primary cells in vitro is slow, the time required to reach the cell number meeting the experimental requirements is long, and it is difficult to maintain a stable physiological state, and they are prone to losing their characteristics and functions. The passage of primary dairy cow follicular granulosa cells can hardly exceed ten generations, and their in vitro culture ability is extremely poor. The existing methods for stable cell passage mainly involve adding cell growth factors to accelerate their growth rate and growth ability by promoting cell proliferation and differentiation, but the number of cell passages still does not change, which seriously limits the research on ovarian function. Therefore, there is an urgent need for a cell model that can be stably passaged and cultured in vitro. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for isolating and culturing ovarian granulosa cells that can be stably passaged and cultured in vitro, so as to solve the problem that existing ovarian granulosa cells cannot be stably passaged in vitro.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a method for separating and culturing ovarian granulosa cells, comprising the following steps: selecting follicles of 3-8 mm in ovarian tissue; collecting follicular fluid by aspiration method and filtering impurities; collecting the filtrate, centrifuging to obtain a precipitate, and obtaining ovarian granulosa cells; resuspending the ovarian granulosa cells with DMEM / F12 complete medium, culturing the cell suspension, and subculturing when the density of ovarian granulosa cells reaches 80%-90%.

[0007] The DMEM / F12 complete medium is composed of the following raw materials according to parts by weight: 80-88 parts of DMEM / F12 medium, 11-19 parts of fetal bovine serum, and 1 part of penicillin-streptomycin mixture.

[0008] Preferably, after obtaining the ovarian tissue, the ovarian tissue is placed in sterile physiological saline containing 2% (v / v) penicillin-streptomycin mixture at 37°C - 39°C.

[0009] Preferably, the aspiration method is to use a syringe to draw DMEM / F12 medium, pierce into the follicle, and then aspirate the liquid in the follicle.

[0010] Preferably, the filtration is carried out using a cell sieve with a pore size of 70 μm.

[0011] Preferably, if the color of the precipitate is not white, the precipitate is resuspended with erythrocyte lysis mixture, allowed to stand and mixed evenly, centrifuged, and the liquid is discarded; the erythrocyte lysis mixture is composed of erythrocyte lysate and DMEM / F12 complete medium according to a volume ratio of 1:2.

[0012] Preferably, the centrifugation speed is 800 rpm / min, the centrifugation time is 6 min; the culture temperature is 37°C.

[0013] Preferably, the subculture includes the following steps: when the density of ovarian granulosa cells reaches 80%-90%, discard the culture medium, wash, add trypsin for digestion; terminate the digestion with DMEM / F12 complete medium, collect the cells by centrifugation; discard the supernatant, resuspend the cells with DMEM / F12 complete medium for culture; change the culture medium every other day; when the density of ovarian granulosa cells reaches 80%-90%, carry out subculture.

[0014] Preferably, it further includes the steps of cell cryopreservation and resuscitation. The cryopreservation solution is composed of fetal bovine serum and DMSO according to a volume ratio of 9:1; the cryopreservation process is: room temperature → 4°C for 20 min → -20°C freezer for 30 min → -80°C overnight → liquid nitrogen.

[0015] Preferably, the resuscitation step is: thaw the cryopreserved material at 37°C, and then mix it with DMEM / F12 complete medium at 37°C.

[0016] Preferably, the ovarian tissue is dairy cow ovarian tissue.

[0017] Advantages of the present invention:

[0018] The method for isolating and culturing ovarian granulosa cells provided by the present invention has strong operability, is simple, has few contaminating cells in the extracted ovarian granulosa cells, has a good growth state after passage, a stable physiological state, and an increased number of passages. The ovarian granulosa cells provided by the present invention lay a foundation for subsequent research on the related mechanisms of dairy cow follicle development and granulosa cell molecular regulation, and the provided method for isolating and culturing ovarian granulosa cells can provide a reference for the isolation and culture of granulosa cells of other mammals. Description of the Drawings

[0019] Figure 1 It is the identification result of the dairy cow ovarian granulosa cell marker gene FSHR by immunofluorescence method. From left to right, they are respectively: the DAPI staining result of the dairy cow ovarian granulosa cell nucleus, the expression of the dairy cow ovarian granulosa cell marker gene FSHR, and the fusion of the two images;

[0020] Figure 2 It is the growth morphology diagram of dairy cow ovarian granulosa cells at different culture times. Among them, A is the result at 24 h, B is the result at 48 h, and C is the result at 72 h;

[0021] Figure 3 It is the comparison result of the cell morphology diagrams at 24 h after preparing ovarian granulosa cells with or without a 70-μm cell filter sieve. Among them, A is the result without filtering with a 70-μm cell sieve, and B is the result after filtering with a 70-μm cell sieve;

[0022] Figure 4 It is the protein expression results of the ovarian granulosa cell differentiation marker molecules StAR, CYP19A1, and LHCGR at 72 h of culturing cells and after passage to the 3rd generation. Among them, A is the Western blot imaging result, and B is the statistical result of the expression levels of each protein, where ** indicates P < 0.01;

[0023] Figure 5 It is the growth curve of dairy cow ovarian granulosa cells;

[0024] Figure 6 It is the influence of different passage times and different cryopreservation times on the cell viability of dairy cow ovarian granulosa cells. Among them, A is the cell viability result at different passage times, and B is the cell viability result at different cryopreservation times. * indicates P < 0.05, *** indicates P < 0.001, and **** indicates P < 0.0001;

[0025] Figure 7To study the effects of different passage numbers and different cryopreservation times on the expression of proteins related to the proliferation of bovine ovarian granulosa cells, where A shows the Western blot imaging results of different passage numbers, B shows the statistical results of the expression levels of each protein at different passage numbers, C shows the Western blot imaging results of different cryopreservation times, and D shows the statistical results of the expression levels of each protein at different cryopreservation times. Here, * indicates P < 0.05, and ** indicates P < 0.01. Specific implementation method

[0026] The present invention provides a method for isolating and culturing ovarian granulosa cells, which includes the following steps: selecting follicles with a diameter of 3 - 8 mm in ovarian tissue; collecting follicular fluid by aspiration and filtering impurities; collecting the filtrate, centrifuging to obtain the precipitate, and obtaining ovarian granulosa cells; resuspending the ovarian granulosa cells with DMEM / F12 complete medium, culturing the cell suspension, and when the density of ovarian granulosa cells reaches 80% - 90%, performing subculture.

[0027] The DMEM / F12 complete medium is composed of the following raw materials by weight: 80 - 88 parts of DMEM / F12 medium, 11 - 19 parts of fetal bovine serum, and 1 part of penicillin - streptomycin mixture.

[0028] In the present invention, after obtaining the ovarian tissue, the ovarian tissue is placed in sterile physiological saline containing 2% (v / v) penicillin - streptomycin mixture at 37°C - 39°C. Preheating the liquid reagent to 37°C - 39°C can simulate the growth environment temperature in the ovary. In the present invention, all surgical instruments for collecting ovarian tissue are preferably sterilized by high - pressure steam. After obtaining the ovarian tissue, ovarian cell extraction is preferably carried out within 2 hours to reduce the time after the ovary is removed from the body. In the present invention, the ovarian tissue is preferably bovine ovarian tissue. In the present invention, the penicillin content in the penicillin - streptomycin mixture is preferably 10000 U / mL, and the streptomycin content is preferably 10000 μg / mL. In the present invention, the DMEM / F12 complete medium is preferably composed of the following raw materials by weight: 82 - 86 parts of DMEM / F12 medium, 13 - 17 parts of fetal bovine serum, and 1 part of penicillin - streptomycin mixture.

[0029] In the present invention, the aspiration method preferably uses a syringe to draw the DMEM / F12 medium, pierce into the follicles, and then aspirate the liquid in the follicles. The syringe is preferably a 5 mL syringe. The filtration preferably uses a cell sieve with a pore size of 70 μm for filtration. After collecting the filtrate, centrifuge it. The centrifugation conditions are preferably centrifugation at 800 rpm / min for 6 min. The centrifugation conditions defined in the present invention can reduce the damage to primary cells caused by high-speed centrifugation; after centrifugation, take the precipitate. Since the ovaries are rich in blood vessels, although the aspiration method can simplify the collection step, the probability of piercing blood vessels and mixing in red blood cells is increased during the operation. Therefore, if the color of the precipitate is not white, it is preferably necessary to resuspend the precipitate with a red blood cell lysis mixture, let it stand and mix well, centrifuge, discard the liquid, and the precipitate taken is ovarian granulosa cells; the red blood cell lysis mixture is composed of a red blood cell lysate and DMEM / F12 complete medium in a volume ratio of 1:2. The present invention has no special limitation on the specific sources of the raw materials in the red blood cell lysate and DMEM / F12 complete medium, and conventional commercially available products in the art can be used.

[0030] In the present invention, the culture temperature is preferably 37 °C. During primary culture, it is preferred to change the medium after culturing for 48 h. Observe the cell attachment and growth status during the culture period. The cells grow in a monolayer and are routinely passaged two to three times a week.

[0031] In the present invention, the subculture preferably includes the following steps: when the density of ovarian granulosa cells reaches 80% - 90%, discard the culture medium, wash it, and then add trypsin for digestion; after terminating the digestion with DMEM / F12 complete medium, collect the cells and centrifuge; after discarding the supernatant, resuspend the cells with DMEM / F12 complete medium for culture; change the culture medium every other day; when the density of ovarian granulosa cells reaches 80% - 90%, perform subculture. In the subculture described in the present invention, the washing is preferably performed with sterile PBS; the digestion conditions are preferably digestion at 37 °C for 2 min.

[0032] In the present invention, preferably, it further includes the steps of cell cryopreservation and resuscitation. The cryopreservation solution for cryopreservation is composed of fetal bovine serum and DMSO at a volume ratio of 9:1. The preferred cryopreservation process is: room temperature → 4°C for 20 min → freezer of -20°C refrigerator for 30 min → -80°C overnight → liquid nitrogen. In the present invention, the preferred resuscitation step is: thaw the cryopreserved material at 37°C, and then mix it with the complete DMEM / F12 medium at 37°C. The method for isolating and culturing ovarian granulosa cells provided by the present invention is improved in aspects such as tissue collection, cell extraction, culture conditions, cell passage, cell cryopreservation, and cell resuscitation, and thus primary dairy cow follicular granulosa cells with excellent growth and development conditions are extracted, which can be stably passaged and cultured in vitro. In addition, the present invention takes the ovarian tissue of dairy cows in the estrous cycle as the research object to establish a system for isolating and culturing ovarian granulosa cells of dairy cows, identifies the isolated cells by immunofluorescence, and determines the passage times and cryopreservation time for dairy cow follicular granulosa cells to maintain the best proliferation activity and growth vitality through methods such as MTS and Western blot, which can provide a research basis for the molecular regulation mechanism of dairy cow ovarian granulosa cells and the cell model of hormone metabolism, and provide a reference for the isolation and culture of ovarian granulosa cells of other mammals.

[0033] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] In the following embodiments, unless otherwise specified, all are conventional methods.

[0035] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0036] The DMEM / F12 medium (SH30023.01) in the following embodiments was purchased from HyClone; the primary antibodies against CCND1, CCND2, CCND3, CCNE1, CDK2, CDK4, CDK6, LHCGR, CYP19A1, StAR, β-actin, and the secondary antibody labeled with HRP were purchased from Proteintech; PBS buffer, dimethyl sulfoxide (DMSO), SDS-PAGE gel preparation kit were purchased from Solarbio (Beijing); penicillin-streptomycin mixture was purchased from Beyotime; fetal bovine serum (FBS) was purchased from CellMax (Australia); trypsin was purchased from Gibco (USA); 4% paraformaldehyde was purchased from Boster; protein loading buffer was purchased from Boster; MTS cell proliferation detection kit and PI / RNase staining buffer were purchased from Beyotime Biotechnology Company.

[0037] In the following examples, the DMEM / F12 complete medium consists of 84 parts of DMEM / F12 basal medium, 15 parts of fetal bovine serum, and 1 part of penicillin-streptomycin mixture (in the penicillin-streptomycin mixture, the penicillin content is 10,000 U / mL and the streptomycin content is 10,000 μg / mL).

[0038] In the penicillin-streptomycin mixture in the following examples, the penicillin content is 10,000 U / mL and the streptomycin content is 10,000 μg / mL.

[0039] Example 1

[0040] (1) Tissue collection method:

[0041] Sample source: from a slaughterhouse in Longfeng District, Daqing City, Heilongjiang Province.

[0042] Pretreatment of reagents and consumables required for sampling: Keep-warm buckets, surgical scissors, surgical forceps, and 1 L beakers are sterilized by high-pressure steam in advance; 2 L of 38°C sterile physiological saline containing 2% penicillin-streptomycin mixture (Beyotime, China, penicillin content: 10,000 U / mL, streptomycin content: 10,000 μg / mL) is placed in the keep-warm bucket; 38°C sterile physiological saline.

[0043] Sample collection method: First, spray and disinfect the collection site with alcohol, collect the bilateral ovaries of dairy cows with sterile surgical instruments, cut off the surrounding redundant tissues, rinse the impurities and blood on the surface of the ovaries with 38°C sterile physiological saline containing 2% penicillin-streptomycin mixture, and place the ovaries in the keep-warm bucket and bring them back to the laboratory of Heilongjiang Bayi Agricultural University within 2 h.

[0044] (2) Collection of follicular fluid

[0045] Place the ovaries in a beaker and rinse them with 38°C sterile physiological saline containing 2% penicillin-streptomycin mixture until the liquid is clear. After drying the liquid on the surface of the ovaries with sterile gauze, draw 1 mL of DMEM / F12 medium with a 5 mL syringe and pierce into follicles with a diameter of 3 - 8 mm. Aspirate the follicular fluid and mix it with the DMEM / F12 medium in the syringe, and collect the mixture of follicular fluid and DMEM / F12 medium into a 50 mL centrifuge tube. Filter out impurities with a cell sieve with a pore size of 70 μm.

[0046] (3) Preparation of primary ovarian granulosa cells

[0047] Centrifuge the follicular fluid at 800 rpm / min for 6 min and take the precipitate. If the precipitate is not a white cell precipitate, prepare a mixed solution according to the ratio of red blood cell lysate: DMEM / F12 complete medium = 1:2, take 5 mL to resuspend the centrifuged precipitate, let it stand for 10 min, gently vortex and mix twice during this period to remove the red blood cells introduced during the collection of follicular fluid, and then centrifuge at 800 rpm / min for 6 min. Subsequently, wash the cell precipitate with 1 mL of sterile PBS buffer containing 1% penicillin-streptomycin mixture at 38°C, centrifuge and discard the supernatant. This process of washing the cell precipitate and centrifuging and discarding the supernatant is repeated 5 times. The cell precipitate is the primary ovarian granulosa cells of dairy cows.

[0048] According to the characteristic of the specific expression of Follicle-Stimulating Hormone Receptor (FSHR) in ovarian granulosa cells, use immunofluorescence to detect the expression of FSHR in the extracted cells and identify the purity of granulosa cells; the specific operation steps of immunofluorescence are as follows:

[0049] a. Cell preparation: When subculturing ovarian granulosa cells, place cell slides in six-well plates in advance, inoculate the cell suspension at 1×10 5 cells / mL into six-well plates. When the cell confluence reaches 80%-90%, wash with PBS buffer 3 times, 5 min each time;

[0050] b. Fixation: Fix with 4% paraformaldehyde for 30 min, wash with PBS buffer 3 times, 5 min each time;

[0051] c. Permeabilization: Perform permeabilization treatment on the fixed cells, incubate with 0.3% Triton X-100 on ice for 15 min to ensure that the antibody can reach the antigen epitope, and then wash with PBS buffer 3 times, 5 min each time;

[0052] d. Blocking: Incubate with blocking solution at room temperature (0.5% fetal bovine serum + 0.3% Triton X-100) for 1 h, wash with PBS buffer 3 times, 5 min each time;

[0053] e. Primary antibody binding: Add primary antibody (anti-FSHR, 1:500), incubate overnight at 4°C, wash with PBS buffer 3 times, 5 min each time;

[0054] f. Secondary antibody binding: Add secondary antibody (FITC, 1:500), incubate for 1 h at room temperature in the dark;

[0055] g. Wash with PBS buffer 3 times, 5 min each time, add DAPI for nuclear staining for 10 min;

[0056] h. Observe the expression of FSHR under a fluorescence microscope and take pictures for retention.

[0057] The results are as Figure 1 shown. After DAPI staining, the cell nuclei appear blue, and the receptor protein FSHR is red and located in the cytoplasm, indicating that the granulosa cells extracted according to the above method are bovine ovarian granulosa cells.

[0058] (4) Primary culture

[0059] Resuspend the cell pellet with DMEM / F12 complete medium and inoculate the cell suspension into a T25 cell culture flask. Place it in a carbon dioxide incubator and culture at 37°C for 48 h, then change the medium. Observe the cell attachment and growth status during this period. The cells grow in a monolayer and are routinely passaged two to three times a week.

[0060] (5) Subculture and amplification

[0061] When the density of ovarian granulosa cells reaches 80 - 90%, discard the culture medium. After washing with sterile PBS, add 2 mL of trypsin and digest at 37°C for 2 min. After terminating the digestion with 4 mL of DMEM / F12 complete medium, collect the cells and centrifuge at 800 rpm / min for 6 min. After discarding the supernatant, resuspend the cells with DMEM / F12 complete medium and inoculate them into a 60 mm culture dish and a T25 cell culture flask. Observe the cell density under the microscope and change the culture medium the next day. Passage the cells when the confluence reaches 80 - 90%.

[0062] The growth morphology diagrams of bovine ovarian granulosa cells at different culture stages are as Figure 2 shown. It can be seen that after 24 h, the primary ovarian granulosa cells attach and grow with a stable state. After 48 h, the cell confluence reaches 50 - 60%. After 72 h, the cell confluence reaches 80 - 90% and subculture can be carried out.

[0063] (6) Cell cryopreservation and recovery

[0064] Prepare the cell cryopreservation solution according to the ratio of fetal bovine serum: DMSO = 9:1. Select cells in the logarithmic growth phase with a confluence of 80 - 90% for cryopreservation and change the medium one day before cryopreservation. Collect the cultured cells according to the subculture operation, resuspend the cell pellet with the cell cryopreservation solution, and aliquot them into cell cryopreservation tubes and place them in a cryopreservation box. Preserve the cells by the gradient cooling method (room temperature → 4°C (20 min) → freezer of - 20°C refrigerator (30 min) → ultra - low temperature refrigerator (-80°C overnight) → liquid nitrogen). Mark the cell type, cell passage number, and cryopreservation date with a marker pen.

[0065] Add 9 mL of pre-warmed 37°C DMEM / F12 complete medium to a 15 mL centrifuge tube. Take out the cell cryopreservation tube from liquid nitrogen, quickly place one end in a 37°C constant temperature water bath and gently shake the tube until the cryopreservation solution melts, then stop the resuscitation. Add the cryopreservation solution to the previously prepared centrifuge tube containing 37°C DMEM / F12 complete medium, centrifuge at 800 rpm / min for 6 min, discard the supernatant, take 1 mL of DMEM / F12 complete medium to resuspend the cell pellet, place the cell suspension in a T25 cell culture flask, and culture it in a 37°C constant temperature incubator.

[0066] Example 2

[0067] The difference from Example 1 is that in step (2), a 70 μm cell sieve is not used for filtration, and the rest is the same as in Example 1.

[0068] After obtaining ovarian granulosa cells by the methods of Example 1 and this Example 2 respectively and culturing them for 24 h, observe the different cell morphologies. The results are as Figure 3 shown. After filtration with a cell sieve, the amount of miscellaneous cells and tissue fragments is relatively reduced, reducing the interference conditions and influencing factors for the primary culture of ovarian granulosa cells.

[0069] Example 3

[0070] After obtaining ovarian granulosa cells by the method of Example 1, culture the cells for 72 h, and passage them to the 3rd generation (F3) according to the method of Example 1. Use Western blot to detect the protein expression changes of the differentiation marker molecules steroidogenic acute regulatory protein (StAR), aromatase (CYP19A1), and luteinizing hormone / choriogonadotropin receptor (LHCGR) when the ovarian granulosa cells are cultured for 72 h and passaged to the 3rd generation.

[0071] The specific operation steps of Western blot are as follows:

[0072] Cells at different culture times were collected to extract total proteins, and the protein concentration was measured by the BCA method (the BCA kit was purchased from Beyotime). Protein samples were dripped onto an SDS-PAGE gel for electrophoresis and transferred to a PVDF membrane (total protein concentration was 20 μg). The membrane was blocked with 5% non-fat milk solution at room temperature for 1.5 h, and then incubated overnight at 4°C with the following primary antibodies: StAR (1:2000; Proteintech), CYP19A (1:1000; Proteintech), and LHCGR (1:1000; Proteintech); β-actin (1:10000; Proteintech). The primary antibodies were washed 5 times with TBST, 6 min each time. The HRP-labeled secondary antibody (1:10000; Proteintech) was incubated at room temperature for 1 h, and the secondary antibody was washed 5 times with TBST, 6 min each time. Bands were visualized using an enhanced chemiluminescence substrate (Thermo Fischer Scientific) and imaged in a multifunctional imaging system. Image analysis was performed using Image Pro Plus 5.0 software.

[0073] The results are as Figure 4 shown, indicating that the extracted ovarian granulosa cells had strong differentiation ability when cultured to the third generation.

[0074] Example 4

[0075] Calculate the population doubling time and plot the growth curve of ovarian granulosa cells

[0076] The dairy cow ovarian granulosa cells prepared according to the method of Example 1 were evenly seeded in a 24-well plate at a density of 2×10 4 / well, and the medium was changed daily. At the same time every day, 3 wells of cells were taken, digested with trypsin, counted, and the average value was taken. The growth curve was plotted with the culture time as the abscissa and the cell quantity as the ordinate, and the population doubling time (PDT) was calculated. The calculation formula is as follows: PDT = [lg2 / (lgNt - lgN0)] × t, where Nt = the number of cells after culture, N0 = the number of seeded cells, and t is the culture time.

[0077] The results are as Figure 5 shown. 0 - 24 h: The cells were in the adherent adaptation period, and there was almost no increase in cell number. 24 - 48 h: The cell number increased slightly, and the growth rate was slow. 48 - 120 h: The cells divided vigorously during this period, and the cell number increased significantly. At 120 h, the cell number reached the peak. After 120 h: The cell number decreased slightly. The population doubling time of ovarian granulosa cells from 0 to 120 h was: 37.3 h.

[0078] Example 5

[0079] MTS assay was used to detect the effects of different passage times and cryopreservation time on the cell viability and proliferation ability of ovarian granulosa cells

[0080] According to the method of Example 1, the cells were passaged for 1 (F1), 3 (F3), 6 (F6) and 12 (F12) generations respectively. According to the method of Example 1, the cells were frozen for 30 days, 90 days and 180 days respectively and then revived. The effects of different passage numbers and freezing times on the proliferation activity of ovarian granulosa cells were detected according to the instructions of the MTS kit.

[0081] Cells of different passage times were counted as 1×10 5 The cells were inoculated at a density of 100 / mL in a 96-well plate. After culturing for 24 hours, 20 μL MTS reagent was added to each well of cells and placed in an incubator for 4 hours. The absorbance at 490 nm was detected using an ELISA reader. Six replicate wells were set up for each group, and each group of experiments was repeated at least 3 times. The results are shown in the figure. Figure 6 As shown in A, the cells passaged to the third generation have a strong proliferation ability, and the cell activity gradually decreases with the increase in the number of passages.

[0082] Using the same experimental method, ovarian granulosa cells that were cryopreserved for 30 days, 90 days, and 180 days were revived and cultured at 1×10 5 The cells were inoculated at a density of 100 / mL in a 96-well plate. After culturing for 24 hours, 20 μL MTS reagent was added to each well of cells and placed in an incubator for 4 hours. The absorbance at 490 nm was detected using an ELISA reader. Six replicate wells were set up for each group, and each group of experiments was repeated at least 3 times. The results are shown in the figure. Figure 6 As shown in B, the proliferation ability of cells decreased with the extension of freezing time.

[0083] Example 6

[0084] Western blot was used to detect the effects of different passage times and freezing time of bovine ovarian granulosa cells on the expression of cell proliferation-related proteins.

[0085] Passage 1 (F1), 3 (F3), 6 (F6) and 12 (F12) were carried out respectively according to the method of Example 1. After cryopreservation for 30 days, 90 days and 180 days and then resuscitation according to the method of Example 1, Western blot detection was performed. The specific method of Western blot detection was the same as that of Example 3, except that the following primary antibodies were incubated overnight at 4°C: CCND1 (1:3000; Proteintech), CCND2 (1:2000; Proteintech), CCND3 (1:2000; Proteintech), CCNE1 (1:3000; Proteintech), CDK2 (1:2000; Proteintech), CDK4 (1:3000; Proteintech), CDK6 (1:2000; Proteintech).

[0086] The results are as Figure 7 shown. The protein expressions of cyclins (CCND1, CCND2, CCND3, CCNE1) and cyclin-dependent kinases (CDK2, CDK4, CDK6) in ovarian granulosa cells passaged to the 3rd generation were significantly up-regulated, indicating that the proliferation activity of ovarian granulosa cells passaged to the 3rd generation was stronger; with the extension of cryopreservation time, the protein expressions of cyclins (CCND1, CCND2, CCND3, CCNE1) and cyclin-dependent kinases (CDK2, CDK4, CDK6) decreased.

[0087] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for isolating and culturing ovarian granulosa cells, characterized in that: The method comprises the following steps: selecting follicles of 3 to 8 mm in ovarian tissue; collecting follicular fluid by suction and filtering impurities; collecting the filtrate and centrifuging to obtain precipitate to obtain ovarian granulosa cells; resuspending the ovarian granulosa cells with DMEM / F12 complete culture medium, culturing the cell suspension, and subculturing when the density of the ovarian granulosa cells reaches 80% to 90%; The DMEM / F12 complete culture medium is composed of the following raw materials in parts by weight: 80-88 parts of DMEM / F12 culture medium, 11-19 parts of fetal bovine serum and 1 part of penicillin-streptomycin mixed solution.

2. The method according to claim 1, characterized in that After the ovarian tissue is collected, it is placed in a sterile physiological saline solution containing 2% by volume of a penicillin-streptomycin mixture at 37°C to 39°C.

3. The method according to claim 1, characterized in that The aspiration method is to use a syringe to extract DMEM / F12 culture medium, pierce the follicle, and then aspirate the liquid in the follicle.

4. The method according to claim 1, characterized in that: The filtration is carried out using a cell sieve with a pore size of 70 μm.

5. The method according to claim 1, characterized in that If the color of the precipitate is not white, resuspend the precipitate with a red blood cell lysis mixture, let it stand to mix, centrifuge, and discard the liquid; the red blood cell lysis mixture consists of a red blood cell lysis solution and a DMEM / F12 complete culture medium in a volume ratio of 1:

2.

6. The method according to claim 1, characterized in that The centrifugal speed is 800 rpm / min, the centrifugal time is 6 min; and the culture temperature is 37°C.

7. The method according to claim 1, characterized in that The subculture comprises the following steps: when the density of the ovarian granulosa cells reaches 80% to 90%, discarding the culture medium, washing, adding trypsin for digestion; terminating the digestion with DMEM / F12 complete culture medium, collecting the cells for centrifugation; discarding the supernatant, resuspending the cells with DMEM / F12 complete culture medium for culture; replacing the culture medium every other day; and when the density of the ovarian granulosa cells reaches 80% to 90%, subculture is performed.

8. The method according to claim 1, characterized in that The method also includes the steps of cell freezing and thawing, wherein the freezing solution is composed of fetal bovine serum and DMSO in a volume ratio of 9:1; the freezing process is: room temperature → 4°C for 20 minutes → -20°C refrigerator freezer for 30 minutes → -80°C overnight → liquid nitrogen.

9. The method according to claim 8, characterized in that The resuscitation step is: thawing the frozen material at 37°C, and then mixing it with 37°C DMEM / F12 complete culture medium.

10. The method according to claim 1, characterized in that The ovarian tissue is cow ovarian tissue.

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