A method for isolating and culturing ovary granulosa cells of Chinese soft-shelled turtle

Abstract

The application discloses a method for separating and culturing ovary granulosa cells of Chinese soft-shelled turtles, which comprises the steps of selecting female Chinese soft-shelled turtles in the positive oviposition period, executing cervical dislocation after anesthesia, killing the turtles, separating ovary tissues, removing redundant parts after washing, separating II-III stage follicles in a basic culture medium, keeping follicle integrity, performing enzyme digestion treatment, removing supernatant through centrifugation, obtaining a precipitate containing ovules with a diameter of 0.1-0.5 cm, resuspending the precipitate by using a mixed culture medium, and inoculating and culturing. The ovary granulosa cells obtained by the separation method have a high yield, the ovary granulosa cells are cultured in the specific mixed culture medium, the method can replace many factors, the amount of added factors is small, the NaHCO3-free culture medium is used, the use of CO2 is reduced, and the ovary granulosa cells obtained are consistent with the in-vivo environment, so that the cost is reduced and the in-vitro survival period of the ovary granulosa cells is prolonged.

Description

Technical Field

[0001] This invention relates to the field of cell biology engineering technology, specifically to a method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle. Background Technology

[0002] In modern turtle and tortoise farming, due to the depletion of wild resources, artificial breeding faces problems such as asynchronous gonadal development and low egg production. Furthermore, follicle development depends on the stability and interaction between ovarian granulosa cells and oocytes. Ovarian cells in the Chinese soft-shelled turtle (Typhonus sinensis) are a collective term for all cells in the ovarian tissue, a mixed population of various cell types, while ovarian granulosa cells are a single type of specialized cell. Differences in their specific composition directly affect the difficulty of experimental isolation. As the main somatic cell group in the Chinese soft-shelled turtle follicles, ovarian granulosa cells not only provide nutrients and transmit signals for oocyte development but also participate in physiological functions such as follicle development, hormone secretion (e.g., estrogen, anti-Müllerian hormone AMH), and regulation of oocyte development and differentiation. Currently, primary isolation techniques for ovarian granulosa cells are relatively mature, but most studies focus on mammals and fish; in vitro culture of Chinese soft-shelled turtle ovarian granulosa cells is rarely reported. Moreover, primary cells are difficult to passage in vitro for long periods; as the number of passages increases, cell growth rate and morphology are gradually affected, failing to meet experimental requirements. Furthermore, the process of re-obtaining ovarian granulosa cells is cumbersome and significantly increases costs. Therefore, how to successfully invent a method for isolating and culturing Chinese soft-shelled turtle ovarian granulosa cells has become a technical bottleneck that urgently needs to be overcome.

[0003] Compared to other species, the Chinese soft-shelled turtle's follicles consist of only a single layer of granulosa cells surrounding the oocyte. These cells are small, with a diameter of only 8-12 μm. Due to the sparse distribution of follicles and the thin granulosa layer, the density of granulosa cells per unit ovarian tissue is low, making them easily lost during separation and leading to cell separation failure. As a poikilothermic animal, the Chinese soft-shelled turtle lacks a thermoregulatory mechanism in its cells. The tissue processing and cell collection procedures during separation can easily exacerbate the stress response of the cells, resulting in unstable survival rates of the separated granulosa cells. Furthermore, the separated granulosa cells may experience decreased activity and mass apoptosis due to temperature stress. Summary of the Invention

[0004] The purpose of this invention is to provide a simple, stable and efficient method for isolating and culturing Chinese soft-shelled turtle ovarian cells, laying a theoretical foundation and providing scientific and technological support for the subsequent development of Chinese soft-shelled turtle follicles.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for isolating and culturing granulosa cells from the ovaries of Chinese soft-shelled turtles, comprising isolation and culture, characterized in that: the isolation involves selecting female Chinese soft-shelled turtles in their egg-laying period, euthanizing them by dislocating their necks after anesthesia, separating the ovarian tissue, cleaning and removing excess tissue, then placing it in a basal culture medium to isolate stage II-III follicles while maintaining follicle integrity, performing enzymatic digestion, then centrifuging to remove the supernatant, obtaining a precipitate containing eggs with a diameter of 0.1-0.5 cm, resuspending the precipitate in a mixed culture medium, and inoculating for culture.

[0007] The eggs with a diameter of 0.1-0.5 cm from the female Chinese softshell turtles during the egg-laying period are obtained from the II-III stage follicles of sexually mature individuals. Long-term experiments have shown that if the obtained eggs are not from II-III stage follicles, the survival rate of the corresponding granulosa cells isolated is extremely low.

[0008] Furthermore, the basal culture medium is a NaHCO3-free DMEM medium, with each 1000 mL of the culture medium system containing 4.2~4.8 g / L D-glucose, 0.2~0.3 g / L L-glutamine, 105~115 mg / L sodium pyruvate and 7.5~7.7 g / L HEPES.

[0009] Furthermore, the enzyme digestion involves first performing a first-step enzyme digestion using collagenase IV for 18-22 minutes, followed by a second-step enzyme digestion using a complex enzyme consisting of collagenase IV and trypsin for 8-12 minutes.

[0010] Furthermore, the concentration of collagenase IV used in the first step of enzymatic digestion is 0.8~1.5 mg / mL. Enzymatic digestion removes external connective tissue and vascular remnants from the ovarian tissue. In the second step of enzymatic digestion, the ovarian tissue after the first step of enzymatic digestion is digested with a mixed enzyme consisting of 1 mg / mL collagenase IV and 0.25% trypsin in a 1:1 volume ratio to eliminate surface contaminant cells, expose the target cells, and maintain a constant temperature of 28°C.

[0011] The digestion time needs to be strictly controlled during this process, as changes in digestion time can significantly affect the survival rate of the isolated target cells or increase the difficulty of subsequent separation.

[0012] The follicles of the Chinese soft-shelled turtle are surrounded by only a single layer of granulosa cells. The cells are small, with a diameter of only 8-12 μm. Due to the sparse distribution of follicles and the thin granulosa cell layer, the density of granulosa cells per unit ovarian tissue is low. When isolating ovarian granulosa cells, they are easily lost, leading to isolation failure. Moreover, the isolated ovarian granulosa cells have poor survival ability and do not have the ability to be stably passaged and cultured.

[0013] This invention utilizes follicles at specific stages, maintaining their integrity, combined with a specific two-step enzymatic digestion process to efficiently isolate and obtain granulosa cells from the ovaries of the Chinese soft-shelled turtle. After culturing in a mixed culture medium, the isolated ovarian granulosa cells exhibit excellent survival, proliferation, and stable passage ability. In the art, sodium bicarbonate is typically introduced into the culture medium during cell culture to maintain the acid-base balance of the cell culture medium by buffering pH changes through CO2. However, this study found that the survival and proliferation of Chinese soft-shelled turtle ovarian granulosa cells were significantly inhibited in a CO2-containing culture system, while their survival and proliferation were significantly improved in a CO2-free culture system. Furthermore, adding a trace amount of Chinese soft-shelled turtle serum combined with fetal bovine serum to the culture medium effectively enhanced the proliferation of granulosa cells.

[0014] Furthermore, the mixed culture medium is prepared by adding 15% fetal bovine serum, 1×Pen-Strep, 0.8~1.2% Chinese soft-shelled turtle serum, and growth factors to the above-mentioned basic culture medium. The growth factors are 8~12 ng / mL IGF-1 or EGF.

[0015] Furthermore, the initial medium change time for cell seeding culture is 5 days, and thereafter the mixed medium is changed every 48-72 hours, with the culture temperature kept constant at 28℃.

[0016] Furthermore, after the cells are seeded and cultured, they are passaged using a mixed culture medium. The cells are passaged every 48-72 hours, and the passages are digested with 0.25% trypsin.

[0017] Furthermore, the isolated granulocytes were cryopreserved using a cryopreservation solution composed of dimethyl sulfoxide (DMSO) and bovine serum in a volume ratio of 3-4:16-17. The cells were first pre-cooled at 4°C for 1 hour, then transferred to -20°C for 5 hours, then transferred to -80°C for overnight freezing, and finally stored in liquid nitrogen for long-term preservation.

[0018] Furthermore, the resuscitation process involves rapidly thawing cells in a 28°C constant temperature water bath, centrifuging to remove the cryopreservation solution, and then inoculating and culturing the resuscitated cells.

[0019] Specifically, a method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle is characterized by comprising the following steps:

[0020] (1) Select female Chinese softshell turtles weighing 1-2 kg during the egg-laying period. After anesthetizing them with MS222 and euthanizing them by neck dissection, remove the ovarian tissue, wash it with 1×PBS, and remove the excess part.

[0021] (2) Isolate stage II-III follicles in the basal culture medium, keep the follicles intact, perform enzymatic digestion, then centrifuge and discard the supernatant, collect the precipitate. The enzymatic digestion is carried out at a constant temperature of 28°C. First, add 0.8~1.2 mg / mL collagenase IV to digest for 18~22 min, then add mixed enzyme (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) to digest for 8~12 min. The basal culture medium is DMEM medium without NaHCO3. The components of the culture medium system per 1000 mL are 4.2~4.8 g / L D-glucose, 0.2~0.3 g / L L-glutamine, 105~115 mg / L sodium pyruvate and 7.5~7.7 g / L HEPES;

[0022] (3) The precipitate was resuspended in a mixed culture medium and inoculated. The first medium change was 5 days later, and the mixed culture medium was changed every 48-72 hours thereafter. The mixed culture medium was made by adding 15% fetal bovine serum, 1×Pen-Strep, 0.8-1.2% Chinese soft-shelled turtle serum and growth factors to the above basic culture medium. The growth factors were 8-12 ng / mL IGF-1 or EGF.

[0023] The present invention has the following technical effects:

[0024] This invention provides a two-step enzymatic hydrolysis method for obtaining ovarian granulosa cells of the Chinese soft-shelled turtle, which is not only simple to operate and quick, but also yields a high rate of ovarian granulosa cells.

[0025] This invention also provides a method for culturing ovarian granulosa cells in a mixed culture medium, which can replace various factors, requires less factor addition, and uses a NaHCO3-free culture medium to reduce CO2 usage, while maintaining the consistency of the obtained ovarian granulosa cells with the biological characteristics of cells in vivo. This not only reduces costs but also increases the in vitro survival time of ovarian granulosa cells. Attached Figure Description

[0026] Figure 1 HE staining image of Chinese soft-shelled turtle ovarian tissue. B is a magnified view of the rectangular frame of A. I-IV represent different stages of follicle development. ※ indicates theca cells; → indicates ovarian granulosa cells.

[0027] Figure 2 Immunofluorescence identification image of granulosa cells from the ovary of the Chinese soft-shelled turtle (Trionyx sinensis).

[0028] Figure 3 Expression analysis of different genes in gonads and isolated cells.

[0029] Figure 4 Morphology of granulosa cells from the ovary of the Chinese soft-shelled turtle at different stages of adherence and growth; A: Morphology of primary cells on day 5; B: Morphology after 150 days of culture.

[0030] Figure 5 Comparison of granulosa cells from the ovaries of the Chinese soft-shelled turtle at different stages and through different isolation methods.

[0031] Figure 6 The effects of CO2 on ovarian granulosa cells.

[0032] Figure 7 Effects of different serum additives on granulosa cells of the Chinese soft-shelled turtle ovary; A: 15% fetal bovine serum + 1% fetal bovine serum, B: 15% fetal bovine serum + 1% Chinese soft-shelled turtle serum, C: 15% fetal bovine serum + 1% bony fish serum.

[0033] Figure 8 Effects of different concentrations of FBS and Chinese soft-shelled turtle serum on the proliferation of ovarian granulosa cells of Chinese soft-shelled turtle.

[0034] Figure 9 : The number of live and dead cells in passaged cells.

[0035] Figure 10 : The number of viable cells in the resuscitation culture. Detailed Implementation

[0036] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0037] The enzyme activity of type IV collagen used in this invention is greater than 160 units / mg (brand: Gibco);

[0038] The enzyme activity of trypsin is greater than 7500 units / mg (brand: Gibco).

[0039] Example 1

[0040] A method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle includes the following steps:

[0041] (1) Select female Chinese softshell turtles weighing 1-2 kg during the egg-laying period. After anesthetizing them with MS222 and euthanizing them by neck dislocation, separate the ovarian tissue, wash it with 1×PBS, and remove the excess part.

[0042] (2) Isolate stage II-III follicles in the basal medium, keep the follicles intact, perform enzymatic digestion, then centrifuge and discard the supernatant, collect the precipitate. The enzymatic digestion is carried out at a constant temperature of 28°C. First, add 1 mg / mL collagenase IV for 20 min, then add a mixed enzyme (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) for 10 min. The basal medium is DMEM medium without NaHCO3. The components of the medium system per 1000 mL are 4.5 g / L D-glucose, 0.25 g / L L-glutamine, 110 mg / L sodium pyruvate and 7.66 g / L HEPES.

[0043] (3) The precipitate was resuspended in a mixed culture medium and inoculated. The first medium change was 5 days later, and the mixed culture medium was changed every 60 hours thereafter. The mixed culture medium was the above basic culture medium with 15% fetal bovine serum, 1×Pen-Strep, 1% Chinese soft-shelled turtle serum and growth factor added. The growth factor was 10 ng / mL IGF-1.

[0044] HE staining of Chinese soft-shelled turtle ovarian tissue as shown Figure 1 As shown in the figure, B is a magnified view of the rectangular frame of A. It can be seen that the granulosa cells of the ovary of the Chinese soft-shelled turtle during the mid-stage of egg production are mainly distributed in stage II-III follicles. No cells are seen in stage IV and above, or only a small number of granulosa cells are seen. Therefore, the target ovarian granulosa cells can only be obtained during this stage.

[0045] FSHR immunofluorescence identification of isolated and cultured Chinese soft-shelled turtle ovarian granulosa cells as follows: Figure 2 As shown, A: Fluorescence imaging cell image; B: DAPI-stained cell nuclei; C: Overlay of A and B. It can be seen that the main isolated cells all express FSHR protein, confirming that the isolated cells are ovarian granulosa cells of the Chinese soft-shelled turtle.

[0046] Expression analysis of different genes in gonads and isolated cells, such as Figure 3 As shown, the testes and ovaries are from Chinese soft-shelled turtle tissue. M represents the marker, and GCs represent isolated granulosa cells from the Chinese soft-shelled turtle ovary. It can be seen that the ovarian granulosa cells express... foxl2 , bmp3 , inhba , not express dnd1 The genetic analysis further confirmed that the isolated cells were ovarian granulosa cells.

[0047] Cell culture at different stages of adherent growth morphology, such as Figure 4 As shown, A is a morphological image of primary cells on day 5, and B is a morphological image of cells cultured for 150 days. It can be seen that after 150 days of culture, the cells adhere well, indicating strong cell viability and intact cell structure, making them suitable for subsequent experiments.

[0048] During the separation process, the unique structure of granulosa cells in the ovaries of the Chinese soft-shelled turtle (Typhonium bifidum) made separation difficult. When using Typhonium bifidum in the egg-laying stage (mid-II-III follicles) for separation, the commonly used follicle puncture method was attempted. Separation was also performed using Typhonium bifidum outside the egg-laying stage (late-stage follicles). The methods of separating follicles by destroying their structure versus maintaining their integrity were also compared. The results are as follows: Figure 5 As shown, A: No puncture of early and mid-stage follicles; B: Puncture of early and mid-stage follicles; C: No puncture of late-stage follicles; D: Puncture of late-stage follicles. It can be seen that a small number of granulosa cells were isolated after puncturing early and mid-stage follicles, but they could not be cultured for a long time. Granulosa cells obtained without puncturing could be cultured stably for a long time. However, no target ovarian granulosa cells were isolated from late-stage follicles, regardless of whether they were punctured or not.

[0049] Comparative Example 1

[0050] Compared with Example 1, the specific enzyme digestion steps are as follows:

[0051] Follicles were collected and digested at a constant temperature of 28°C for 20 minutes with 1 mg / mL collagenase IV, followed by digestion in 0.25% trypsin for 10 minutes.

[0052] Even with the same enzyme concentration and stepwise digestion time, the expected target ovarian granulosa cells were not effectively isolated. This shows that in the stepwise digestion process, the second step of using a complex enzyme is particularly important for obtaining ovarian granulosa cells, based on the first step of using collagenase IV digestion.

[0053] Comparative Example 2:

[0054] The difference from Example 1 is that sodium bicarbonate was added to the culture medium used; otherwise, they are the same as in Example 1.

[0055] The effects of Example 1 (without CO2) and Comparative Example 2 (with CO2) on ovarian granulosa cells are as follows: Figure 6 As shown, granulosa cells in the CO2-treated group exhibited significant cell death and were unable to adhere to the culture medium for growth, while granulosa cells in the CO2-free group showed excellent growth and high cell viability. This demonstrates that the use of a CO2-free medium in the specific mixed culture medium of this invention reduces CO2 usage and maintains the biological characteristics of the obtained ovarian granulosa cells consistent with those in vivo, thereby not only reducing costs but also increasing the in vitro survival time of ovarian granulosa cells.

[0056] Comparative Example 3

[0057] Compared with Example 1, the difference is that the mixture is fully cultured and bony fish serum is used instead of Chinese soft-shelled turtle serum, while the remaining steps are the same as in Example 1.

[0058] The serum added in Example 1 and Comparative Example 3 were different. The effects of adding different types of serum on the granulosa cells of the Chinese soft-shelled turtle ovary are as follows: Figure 7 As shown, A is the control group with 1% fetal bovine serum added to the initial 15% fetal bovine serum, B is the control group with 1% Chinese soft-shelled turtle serum added to the 15% fetal bovine serum in Example 1, and C is the control group with 1% bony fish serum added to the 15% fetal bovine serum in Comparative Example 3. It can be seen that the group of Example 1 (B) is in better condition and has a better growth-promoting effect than the control group (A) with fetal bovine serum alone and the bony fish serum group (C) in Comparative Example 3.

[0059] Furthermore, different concentrations of fetal bovine serum (FBS) in the culture medium, and the further addition of different concentrations of Chinese soft-shelled turtle serum, also showed significant differences in their effects on granulosa cells of the Chinese soft-shelled turtle ovary. For example... Figure 8 As shown, the FBS concentration of 15% resulted in the most stable and optimal overall cell proliferation effect. Further addition of Chinese soft-shelled turtle serum showed that too little serum had an unsatisfactory effect, while too much serum inhibited the proliferation of ovarian granulosa cells. However, a concentration of 1% significantly promoted the proliferation of ovarian granulosa cells.

[0060] Successive generations of cultivation:

[0061] The isolated ovarian granulosa cells were passaged in a mixed culture medium, with passages performed every 48-72 hours. Cells were digested with 0.25% trypsin before passage. The morphology after passage was as follows: Figure 9 As shown in the diagram, the cell number significantly increased after two passages, decreased after the third passage, but the percentage of dead cells also decreased, and remained relatively stable thereafter, with the percentage of dead cells gradually decreasing. This indicates that the ovarian granulosa cells isolated by this invention can be stably passaged, and the survival rate of passaged cells is ≥90%.

[0062] Reviving after freezing:

[0063] (1) The isolated granulocytes were cryopreserved. The cryopreservation solution used was composed of dimethyl sulfoxide (DMSO) and bovine serum in a volume ratio of 5:16. The cells were first frozen at 4°C for 1 hour, then at -20°C for 5 hours, then at -80°C overnight, and finally stored in liquid nitrogen for long-term preservation.

[0064] (2) The cells were revived in a constant temperature water bath at 28°C, centrifuged to remove the cryopreservation solution, and then inoculated with the revived cells.

[0065] As the number of cryopreservation and thawing cycles increases, the cell number changes as follows: Figure 10 As shown, the number of cells gradually increases with the number of cryopreservations, while the percentage of dead cells gradually decreases. This indicates that the ovarian granulosa cells isolated above, after cryopreservation and thawing in a specific culture medium, can maintain a survival rate of ≥80% after thawing, demonstrating excellent in vitro viability and stability, which is beneficial for the long-term preservation and culture of granulosa cells.

[0066] Example 2

[0067] A method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle includes the following steps:

[0068] (1) Select female Chinese softshell turtles weighing 1-2 kg during the egg-laying period. After anesthetizing them with MS222 and euthanizing them by neck dislocation, separate the ovarian tissue, wash it with 1×PBS, and remove the excess part.

[0069] (2) Follicles during the spawning period were isolated from the basal culture medium, and the follicles were kept intact. They were then subjected to enzymatic digestion, centrifuged to discard the supernatant, and the precipitate was collected. The enzymatic digestion was carried out at a constant temperature of 28°C. First, 0.8 mg / mL collagenase IV was added for digestion for 22 min, and then a mixed enzyme (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) was added for digestion for 12 min. The basal culture medium was DMEM medium without NaHCO3. The components of the culture medium system per 1000 mL were 4.2 g / L D-glucose, 0.2 g / L L-glutamine, 105 mg / L sodium pyruvate and 7.7 g / L HEPES.

[0070] (3) The precipitate was resuspended in a mixed culture medium and inoculated. The first medium change was 5 days later, and the mixed culture medium was changed every 48 hours thereafter. The mixed culture medium was the above basic culture medium with 15% fetal bovine serum, 1×Pen-Strep, 0.8% Chinese soft-shelled turtle serum and growth factor added. The growth factor was 8 ng / mL IGF-1.

[0071] The ovarian granulosa cells of the Chinese soft-shelled turtle isolated and cultured in this embodiment can be stably passaged, and the survival rate of passaged culture is ≥90%. After cryopreservation and thawing, the survival rate can still be maintained at ≥80%, which still maintains excellent in vitro survival ability and stability, which is conducive to long-term preservation and culture.

[0072] Example 3

[0073] A method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle includes the following steps:

[0074] (1) Select female Chinese softshell turtles weighing 1-2 kg during the egg-laying period. After anesthetizing them with MS222 and euthanizing them by neck dislocation, separate the ovarian tissue, wash it with PBS, and remove the excess part.

[0075] (2) Follicles during the spawning period were isolated from the basal culture medium, and the follicles were kept intact. They were then subjected to enzymatic digestion, centrifuged to discard the supernatant, and the precipitate was collected. The enzymatic digestion was carried out at a constant temperature of 28°C. First, 1.2 mg / mL collagenase IV was added for digestion for 18 min, and then a mixed enzyme (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) was added for digestion for 8 min. The basal culture medium was DMEM medium without NaHCO3. The components of the culture medium system per 1000 mL were 4.8 g / L D-glucose, 0.3 g / L L-glutamine, 115 mg / L sodium pyruvate and 7.5 g / L HEPES.

[0076] (3) The precipitate was resuspended in a mixed culture medium and inoculated. The first medium change was 5 days later, and the mixed culture medium was changed every 72 hours thereafter. The mixed culture medium was the above basic culture medium with the addition of 15% fetal bovine serum, 1×Pen-Strep, 1.2% Chinese soft-shelled turtle serum and growth factor, the growth factor being 12 ng / mL EGF.

[0077] The ovarian granulosa cells of the Chinese soft-shelled turtle isolated and cultured in this embodiment can be stably passaged, and the survival rate of passaged culture is ≥90%. After cryopreservation and thawing, the survival rate can still be maintained at ≥80%, which still maintains excellent in vitro survival ability and stability, which is conducive to long-term preservation and culture.

Claims

1. A method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle, comprising isolation and culture, characterized in that: The separation process involves selecting female Chinese soft-shelled turtles in their egg-laying period, euthanizing them by dislocation of the neck after anesthesia, separating the ovarian tissue, cleaning and removing excess tissue, and then placing it in a basal culture medium to separate stage II-III follicles while maintaining follicle integrity. Enzymatic digestion is then performed, followed by centrifugation to remove the supernatant, obtaining a precipitate containing eggs with a diameter of 0.1-0.5 cm. The precipitate is resuspended in a mixed culture medium and inoculated. The enzymatic digestion process involves a first step using collagenase IV at a concentration of 0.8-1.5 mg / mL for 18-22 minutes, followed by a second step using a compound enzyme consisting of collagenase IV at a concentration of 1 mg / mL and trypsin at a concentration of 0.25% (v / v) at a volume ratio of 1:1 for 8-12 minutes. The basal culture medium is NaHCO3-free DMEM medium. The mixed culture medium consists of the above basal culture medium supplemented with 15% fetal bovine serum, 1×Pen-Strep, 0.8-1.2% Chinese soft-shelled turtle serum, and growth factors at a concentration of 8-12 ng / mL. IGF-1 or EGF.

2. The method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle as described in claim 1, characterized in that: The basal culture medium contains 4.2-4.8 g / L D-glucose, 0.2-0.3 g / L L-glutamine, 105-115 mg / L sodium pyruvate, and 7.5-7.7 g / L HEPES per 1000 mL of the culture medium system.

3. The method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle as described in claim 2, characterized in that: The inoculation culture medium was changed for the first time after 5 days, and then the mixed culture medium was changed every 48-72 hours thereafter, with the culture temperature kept constant at 28℃.

4. The method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle as described in claim 3, characterized in that: The ovarian tissue was isolated by selecting female Chinese softshell turtles weighing 1-2 kg during their egg-laying period. After being anesthetized with MS222 and euthanized by neck dislocation, the ovarian tissue was isolated, washed with 1xPBS, and excess tissue was removed to obtain the ovarian tissue.

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