Separation and culture method of Chinese softshell turtle ovarian granular cells

Abstract

A Chinese softshell turtle ovarian granular cell separation and culture method comprises separation and culture, the separation comprises the following steps: selecting a female Chinese softshell turtle individual in a positive spawning period, anesthetizing the female Chinese softshell turtle individual, killing the female Chinese softshell turtle individual after neck breaking, separating ovarian tissues, cleaning, removing redundant parts, placing the female Chinese softshell turtle individual in a basal culture medium, separating follicles in II-III periods, keeping the integrity of the follicles, performing enzyme digestion treatment, and culturing the follicles in the basal culture medium. And centrifuging to remove the supernatant to obtain a precipitate containing egg granules with the diameter of 0.1-0.5 cm, resuspending the precipitate by adopting a mixed culture medium, and inoculating and culturing. The ovarian granulosa cells separated by the separation method provided by the invention are high in yield, the method for culturing the ovarian granulosa cells in a specific mixed culture medium is diversified in alternative factors and small in added factor amount, meanwhile, a NaHCO3-free culture medium is used, the use of CO2 is reduced, the obtained ovarian granulosa cells are kept consistent with the internal environment of an organism, the cost is reduced, and the method is suitable for large-scale popularization and application. And the in-vitro lifetime of the ovarian granular cells is prolonged.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell biology engineering, and particularly relates to a method for separating and culturing ovary granular cells of Trionyx sinensis. BACKGROUND

[0002] In modern turtle breeding, due to the decrease of wild resources, there are problems such as asynchronous development of gonads and low egg production in artificial reproduction. At the same time, follicular development depends on the stability and interaction of ovary granular cells-oocytes. The ovary cells of Trionyx sinensis are the total of all cells in the ovary tissue, which is a mixed population of various cells, while the ovary granular cells are a single type of specialized cells, and the difference in specific composition directly affects the difficulty of experimental separation. As the main somatic cell population in the follicle of Trionyx sinensis, the ovary granular cells not only provide nutrients and transmit signals for oocyte development, but also participate in follicular development, hormone secretion (such as estrogen and anti-Mullerian hormone AMH) and regulation of oocyte development and differentiation. At present, the primary separation technology of ovary granular cells has been relatively mature, but most of them are concentrated in mammals and fish, and there are few reports on the in vitro culture of ovary granular cells of Trionyx sinensis. At the same time, primary cells are difficult to be cultured in vitro for a long time, and with the increase of the number of generations, the growth rate and morphology of the cells will also be gradually affected, which cannot meet the experimental needs. The steps of obtaining ovary granular cells again are complicated, and the cost is greatly increased. Therefore, how to successfully invent a method for separating and culturing ovary granular cells of Trionyx sinensis has become a technical bottleneck that needs to be broken through.

[0003] Compared with other species, the follicle of Trionyx sinensis only has a single layer of granular cells surrounding the oocyte, and the cell volume is small, with a diameter of only 8-12 μm. Due to the sparse distribution of follicles and the thin granular layer, the granular cell density of unit ovary tissue is low, so that the cells are easily lost during the separation of ovary granular cells, resulting in the failure of cell separation. As a poikilothermic animal, the cells of Trionyx sinensis have no constant temperature regulation mechanism, and the stress response of cells is easily intensified during the process of tissue processing and cell collection operation, resulting in unstable survival rate of granular cells after separation. The activity of granular cells after separation is decreased due to temperature stress, and a large number of apoptosis occurs. SUMMARY

[0004] The present application aims to provide a simple, stable and efficient method for separating and culturing ovary cells of Trionyx sinensis, which lays a theoretical foundation and technological support for subsequent follicular development of Trionyx sinensis. The present application is achieved by the following technical solutions: The application discloses a method for separating and culturing ovary granular cells of Chinese soft-shelled turtles, and belongs to the field of cell culture.

[0005] The ovum of the female Chinese soft-shelled turtle in the oviposition period has a diameter of 0.1-0.5 cm, and is an ovum separated from a II-III stage follicle in a sexually mature individual. It is found through long-term experiments that if the ovum is not from a II-III stage follicle, the survival rate of the separated granular cells is extremely low.

[0006] Further, the basic culture medium is a DMEM culture medium without NaHCO3, and each 1000 mL of the culture medium system 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.

[0007] Further, the enzyme digestion is first carried out by using collagenase IV for the first step of enzyme digestion, and the digestion time is 18-22 min; then the second step of enzyme digestion is carried out by using a composite enzyme composed of collagenase IV and trypsin, and the enzyme digestion time is 8-12 min.

[0008] Further, the concentration of collagenase IV used in the first step of enzyme digestion is 0.8-1.5 mg / mL, and the enzyme digestion removes the residual connective tissue and blood vessels outside the ovary tissue; the second step of enzyme digestion is carried out by adding 1 mg / mL collagenase IV and 0.25% trypsin to the ovary tissue after the first step of enzyme digestion, and the volume ratio of the two is 1:1, so as to eliminate the surface heterocellular cells, expose the target cells and keep the temperature at 28°C.

[0009] In the process, the digestion time needs to be strictly controlled, and the change of the digestion time will significantly affect the survival rate of the separated target cells or increase the difficulty of subsequent separation.

[0010] The follicle of the Chinese soft-shelled turtle only has a single layer of granular cells surrounding the oocyte, and the cell volume is small, with a diameter of only 8-12 μm. Due to the sparse distribution of follicles and the thin granular cell layer, the granular cell density of the unit ovary tissue is low, and the granular cells are extremely easy to flow away during the separation of the ovary granular cells, leading to separation failure. In addition, the survival ability of the separated ovary granular cells is poor, and the ovary granular cells do not have the ability of stable subculture.

[0011] The present application adopts the ovum follicle of a specific period, and keeps its integrity to combine with the specific two-step enzyme digestion treatment, and efficiently separates and obtains the ovary granular cell of the Chinese soft-shelled turtle. After the ovary granular cell is cultured by the mixed culture medium, the ovary granular cell has excellent survival ability, proliferation ability and stable subculture ability. In the cell culture process in the field, sodium bicarbonate is usually introduced into the culture medium, so that the pH value change in the culture medium system is maintained by CO2 to maintain the acid-base balance of the cell culture medium. However, it is found in the research process that the survival and proliferation of the ovary granular cell of the Chinese soft-shelled turtle in the culture system containing CO2 are obviously inhibited, and the survival ability and the proliferation ability of the ovary granular cell are significantly improved in the culture medium system without CO2. In addition, the addition of a small amount of Chinese soft-shelled turtle serum and fetal bovine serum in the culture medium can effectively improve the proliferation effect of the granular cell.

[0012] Further, the mixed culture medium is obtained by adding 15% fetal bovine serum, 1×Pen-Strep, 0.8~1.2% Chinese soft-shelled turtle serum and growth factors into the above-mentioned basic culture medium, and the growth factors are 8~12 ng / mL IGF-1 or EGF.

[0013] Further, the first medium replacement time of the cell inoculation culture is 5d, and then the mixed culture medium is replaced every 48~72h, and the culture temperature is constant at 28℃.

[0014] Further, after the cell inoculation culture, the subculture is carried out, the mixed culture medium is used for the subculture, and the cell subculture is carried out every 48~72h by using 0.25% trypsin for digestion.

[0015] Further, the isolated granular cell is frozen by using the freezing solution, the freezing solution used for freezing is composed of dimethyl sulfoxide DMSO and bovine serum in a volume ratio of 3~4:16~17, is pre-cooled at 4℃ for 1h, is then transferred to-20℃ for freezing for 5h, is then transferred to-80℃ for freezing overnight, and is finally placed in liquid nitrogen for long-term storage.

[0016] Further, the recovery is carried out by quickly thawing the cells in a 28℃ constant temperature water bath, removing the freezing solution by centrifugation, and inoculating and culturing the recovered cells.

[0017] Specifically, a Chinese soft-shelled turtle ovary granular cell separation and culture method, characterized in that it comprises the following steps: (1) selecting a female Chinese soft-shelled turtle with a weight of 1~2kg in the egg-laying period, killing by cervical dislocation after MS222 anesthesia, dissecting and taking out the ovary tissue, washing with 1×PBS, and removing the excess part; (2) Isolate the II-III stage follicle in the basic medium, keep the follicle intact, and perform enzyme digestion treatment, then centrifuge to discard the supernatant, and collect the precipitate, wherein the enzyme digestion is performed at a constant temperature of 28°C, 0.8-1.2 mg / mL collagenase IV is added first and is digested for 18-22 min, then mixed enzymes (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) are added and are digested for 8-12 min, and the basic medium is DMEM medium without NaHCO3, and the content of each 1000 mL medium system component is 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; (3) The precipitate is resuspended in a mixed medium, and inoculation culture is performed, the first medium replacement time is 5 days, and then the mixed medium is replaced every 48-72 h, wherein the mixed medium is obtained by adding 15% fetal bovine serum, 1x Pen-Strep, 0.8-1.2% Chinese soft-shelled turtle serum and growth factors to the above basic medium, and the growth factors are 8-12 ng / mL IGF-1 or EGF.

[0018] The present application has the following technical effects: The present application provides a method for obtaining Chinese soft-shelled turtle ovary granulosa cells by two-step enzyme digestion, which is simple in operation, short in time consumption, and high in yield of ovary granulosa cells.

[0019] The present application also provides a method for culturing ovary granulosa cells by using a mixed medium, which can replace various factors, has a small amount of added factors, uses a NaHCO3-free medium, reduces the use of CO2, and keeps the obtained ovary granulosa cells consistent with the biological characteristics of cells in the body, thereby reducing the cost and increasing the in-vitro survival period of ovary granulosa cells. BRIEF DESCRIPTION OF DRAWINGS Figure 1 : Chinese soft-shelled turtle ovary tissue HE staining diagram, B is an enlarged diagram of a rectangular frame of A, I-IV are different stages of follicles, and * is follicle membrane cell; → indicates ovary granulosa cell.

[0020] Figure 2 : Chinese soft-shelled turtle ovary granulosa cell FSHR immunofluorescence identification diagram.

[0021] Figure 3 : Expression analysis of different genes in gonads and isolated cells.

[0022] Figure 4 : Different stages of Chinese soft-shelled turtle ovary granulosa cell adhesion growth morphology; A: morphology of primary cells on the 5th day; B: morphology diagram of cells cultured for 150 days.

[0023] Figure 5: The comparison of Chinese soft-shelled turtle ovary granulosa cells in different periods and isolation methods.

[0024] Figure 6 : The effect of CO2 on ovary granulosa cells.

[0025] Figure 7 : The effect of different kinds of serum on Chinese soft-shelled turtle ovary granulosa cells; 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% hard bone fish serum.

[0026] Figure 8 : The effect of different concentrations of FBS and Chinese soft-shelled turtle serum on the proliferation of Chinese soft-shelled turtle ovary granulosa cells.

[0027] Figure 9 : The amount of live and dead cells in subculture cells.

[0028] Figure 10 : The amount of live and dead cells in resuscitation culture cells. DETAILED DESCRIPTION

[0029] The application will be described in detail below by examples, and it is necessary to point out here that the following examples are only used to further illustrate the application, and cannot be understood as limiting the protection scope of the application, and the person skilled in the art can make some non-essential improvements and adjustments to the application according to the above content of the application.

[0030] The enzyme activity of the type IV collagen used in the application is (greater than 160 units / mg) (brand: Gibco); The enzyme activity of the enzyme trypsin is (greater than 7500 units / mg) (brand: Gibco).

[0031] Example 1 A Chinese soft-shelled turtle ovary granulosa cell separation and culture method comprises the following steps: (1) Select female Chinese soft-shelled turtles with a weight of 1-2 kg during the spawning period, and after being anesthetized by MS222 and decapitated, the ovary tissue is separated, washed with 1×PBS, and the excess part is removed; (2) Isolate the follicle of stage II-III in the basic medium, keep the follicle intact, and then centrifuge to discard the supernatant and collect the precipitate, wherein the enzyme digestion is carried out at a constant temperature of 28°C, and 1 mg / mL collagenase IV is added first for 20 min, and then mixed enzymes (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) are added for 10 min, and the basic medium is DMEM medium without NaHCO3, and the content of each 1000 mL medium system component is 4.5 g / L D-glucose, 0.25 g / L L-glutamine, 110 mg / L sodium pyruvate and 7.66 g / L HEPES; (3) Resuspend the precipitate with mixed medium, inoculate and culture, the first medium replacement time is 5 days, and then the mixed medium is replaced every 60 h, wherein the mixed medium is obtained by adding 15% fetal bovine serum, 1×Pen-Strep, 1% Chinese soft-shelled turtle serum and growth factors to the above basic medium, and the growth factors are 10 ng / mL IGF-1.

[0032] The HE staining of the ovary tissue of the Chinese soft-shelled turtle is shown in Figure 1 Fig. A is a rectangular frame enlarged view of B, and it can be seen that the granulosa cells of the Chinese soft-shelled turtle in the middle oogenesis period are mainly distributed in the follicles of stage II-III, and no or a small amount of granulosa cells are seen in the cells of stage IV and above, so that the ovary granulosa cells can only be obtained in this period.

[0033] The FSHR immunofluorescence identification of the ovary granulosa cells of the Chinese soft-shelled turtle isolated and cultured is shown in Figure 2 Fig. A is a fluorescence imaging cell diagram, B is a DAPI-stained cell nucleus, and C is a superimposed diagram of A and B, and it can be seen that the main cells isolated express FSHR protein, and it is determined that the cells obtained are ovary granulosa cells of the Chinese soft-shelled turtle.

[0034] The expression analysis of different genes in the gonad and the isolated cells is shown in Figure 3 Fig. The testis and ovary are tissues of the Chinese soft-shelled turtle, M represents a marker, and GCs represent the isolated ovary granulosa cells of the Chinese soft-shelled turtle, and it can be seen that the ovary granulosa cells express foxl2 、 bmp3 、 inhba , and do not express dnd1 gene, and it is again confirmed that the isolated cells are ovary granulosa cells.

[0035] The adherent growth morphology of the cells in the cell culture process at different periods is shown in Figure 4 Fig. A is the morphology diagram of the primary cells at day 5, and B is the morphology diagram of the cells cultured for 150 days. It can be seen that the adherent growth state of the cells cultured for 150 days is good, which indicates that the cell activity is strong, the cell structure is complete, and the cells can be used for subsequent experiments.

[0036] In the separation process, due to the particularity of the granulosa cell structure in the ovary of Chinese soft-shelled turtle, it is difficult to separate. In the separation process of Chinese soft-shelled turtle in the spawning period (II-III prophase follicle), the follicle puncture method commonly used in the art is tried to separate. In addition, Chinese soft-shelled turtles in non-spawning period (late follicle) are also used for separation. The destruction of follicle structure and the separation of intact follicle are also tried. The results are shown in Figure 5 A: not puncturing prophase follicle method; B: puncturing prophase follicle method; C: not puncturing late follicle method; D: puncturing late follicle method. It can be seen that a small amount of granulosa cells are obtained after puncturing the prophase follicle, but they cannot be cultured for a long time. The granulosa cells obtained without puncturing can be stably cultured for a long time. However, whether puncturing or not puncturing the late follicle, the target ovary granulosa cells cannot be separated.

[0037] Comparative Example 1 Compared with Example 1, the specific enzyme digestion steps are as follows: The follicle is collected at a constant temperature of 28°C. First, 1 mg / mL collagenase IV is added and digested for 20 min, and then 0.25% trypsin is added and digested for 10 min.

[0038] Even if the same enzyme concentration and stepwise enzyme digestion time are maintained, the expected target ovary granulosa cells cannot be effectively separated. It can be seen that in the stepwise enzyme digestion process, the enzyme digestion step of using compound enzyme for enzyme digestion on the basis of using collagenase IV for enzyme digestion in the first step is particularly important for obtaining ovary granulosa cells.

[0039] Comparative Example 2 Compared with Example 1, the difference is that sodium bicarbonate is added to the culture medium, and the rest is the same as Example 1.

[0040] The effects of Example 1 (without CO2) and Comparative Example 2 (with CO2) on ovary granulosa cells are shown in Figure 6 It can be seen that the granulosa cells in the CO2 group died obviously and could not adhere and grow. The granulosa cells in the CO2-free group grew well and had high cell activity. It can be seen that in the specific mixed culture medium of the present application, the CO2-free culture medium is used, the use of CO2 is reduced, and the ovary granulosa cells obtained are consistent with the biological characteristics of the granulosa cells in the body. Not only the cost is reduced, but also the in vitro survival period of ovary granulosa cells is increased.

[0041] Comparative Example 3 Compared with Example 1, the difference is that the mixed complete culture uses hard fish serum instead of Chinese soft-shelled turtle serum, and the rest of the steps is the same as Example 1.

[0042] The serum added in Example 1 and Comparative Example 3 is different, and the effects of adding different kinds of serum on the ovary granulosa cells of Chinese soft-shelled turtles are as shown in Figure 7 A is a control group of further adding 1% fetal bovine serum on the basis of initial 15% fetal bovine serum, B is the corresponding 15% fetal bovine serum of Example 1 plus 1% Chinese soft-shelled turtle serum, and C is the 15% fetal bovine serum of Comparative Example 3 plus 1% hard fish serum, it can be seen that the Example 1 group (B) is better than the single fetal bovine serum control group (A) and the hard fish serum group (C) of Comparative Example 3, and has better growth-promoting effect.

[0043] In addition, there are also significant differences in the effects of different concentrations of fetal bovine serum (FBS) in the culture medium and further addition of different concentrations of Chinese soft-shelled turtle serum on the ovary granulosa cells of Chinese soft-shelled turtles. As shown in Figure 8 The FBS concentration is 15%, the overall cell proliferation effect is the most stable and optimal, on this basis, further addition of Chinese soft-shelled turtle serum can see that the addition amount of Chinese soft-shelled turtle serum is too small and the effect is not ideal, and too much addition will inhibit the proliferation of ovary granulosa cells, and when the concentration is 1%, it significantly promotes the proliferation of ovary granulosa cells.

[0044] Subculture: The separated ovary granulosa cells are subcultured using a mixed culture medium, and the cell subculture is performed every 48-72h using 0.25% trypsin digestion. The morphology after subculture is as shown in Figure 9 It can be seen that the cell number significantly increases at the second subculture, and the cell number decreases at the third subculture, but the proportion of dead cells also decreases, and then it is in a relatively stable state, and the proportion of dead cells gradually decreases. It shows that the separated ovary granulosa cells of the application can be stably subcultured, and the survival rate of subculture is ≥90%.

[0045] Freezing and recovery: (1) The separated granulosa cells are frozen. The freezing solution used for freezing is dimethyl sulfoxide DMSO and bovine serum in a volume ratio of 5:16, first frozen at 4°C for 1h, then frozen at -20°C for 5h, then frozen at -80°C overnight, and finally stored in liquid nitrogen for long-term preservation.

[0046] (2) The recovery is in a 28°C constant temperature water bath, the freezing solution is removed by centrifugation, and the recovered cells are inoculated.

[0047] With the increase of the number of freezing and recovery, the change of the cell number is as shown in Figure 10As shown, it can be seen that with the increase of the number of freeze-thaw cycles, the number of cells gradually increases, and the proportion of dead cells gradually decreases. It shows that the ovarian granulosa cells obtained by the above separation can maintain ≥80% survival rate after freeze-thaw recovery in a specific culture medium, still maintain excellent in vitro survival ability and stability, and are conducive to long-term preservation and culture of granulosa cells.

[0048] Example 2 A method for separating and culturing Chinese soft-shelled turtle ovarian granulosa cells, comprising the following steps: (1) Select female Chinese soft-shelled turtles with a weight of 1-2 kg during the spawning period, and after being killed by cervical dislocation under MS222 anesthesia, separate the ovarian tissue, wash it with 1×PBS, and remove the excess part; (2) Separate the follicles during the spawning period in a basic culture medium, keep the follicles intact, perform enzyme digestion treatment, then centrifuge and discard the supernatant, collect the precipitate, the enzyme digestion is carried out at a constant temperature of 28°C, first add 0.8 mg / mL collagenase IV for 22 min, then add mixed enzymes (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) for 12 min, and the basic culture medium is DMEM culture medium without NaHCO3, and each 1000 mL of culture medium system contains 4.2 g / L D-glucose, 0.2 g / L L-glutamine, 105 mg / L sodium pyruvate and 7.7 g / L HEPES; (3) Resuspend the precipitate with a mixed culture medium, inoculate and culture, the first time of medium replacement is 5 days, and then the mixed culture medium is replaced every 48 hours, the mixed culture medium is obtained by adding 15% fetal bovine serum, 1×Pen-Strep, 0.8% Chinese soft-shelled turtle serum and growth factors to the above basic culture medium, and the growth factors are 8 ng / mL IGF-1.

[0049] The Chinese soft-shelled turtle ovarian granulosa cells separated and cultured in this example can be stably passaged, and the survival rate after subculture is ≥90%, and still maintains excellent in vitro survival ability and stability after freeze-thaw recovery, which is conducive to long-term preservation and culture.

[0050] Example 3 A method for separating and culturing Chinese soft-shelled turtle ovarian granulosa cells, comprising the following steps: (1) Select female Chinese soft-shelled turtles with a weight of 1-2 kg during the spawning period, and after being killed by cervical dislocation under MS222 anesthesia, separate the ovarian tissue, wash it with 1×PBS, and remove the excess part; (2) Isolating follicle at oviposition period in basal medium, keeping follicle intact, and then centrifuging to discard supernatant and collect precipitate, wherein the enzyme digestion is carried out at constant temperature of 28°C, first adding 1.2 mg / mL collagenase IV for 18 min, and then adding mixed enzyme (1 mg / mL collagenase IV: 0.25% trypsin = 1:1) for 8 min, and the basal medium is DMEM medium without NaHCO3, and the content of each 1000 mL medium system component is 4.8 g / L D-glucose, 0.3 g / L L-glutamine, 115 mg / L sodium pyruvate and 7.5 g / L HEPES; (3) Resuspending the precipitate with mixed medium, inoculating and culturing, and the first medium replacement time is 5 d, and then the mixed medium is replaced every 72 h, wherein the mixed medium is obtained by adding 15% fetal bovine serum, 1×Pen-Strep, 1.2% Chinese soft-shelled turtle serum and growth factor into the above basal medium, and the growth factor is 12 ng / mL EGF. The isolated and cultured Chinese soft-shelled turtle ovary granulosa cells in the embodiment can be stably passaged, and the survival rate of the passaged culture is ≥90%, and after being frozen and recovered, the survival rate can still be maintained at ≥80%, and the excellent in-vitro survival ability and stability are still maintained, 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 dislocating their necks after anesthesia, separating the ovarian tissue, cleaning it, removing excess tissue, and then placing it in a basal culture medium to separate stage II-III follicles while maintaining follicle integrity. The follicles are then subjected to enzymatic digestion, followed by centrifugation to remove the supernatant, yielding a precipitate containing eggs with a diameter of 0.1-0.5 cm. The precipitate is resuspended in a mixed culture medium and inoculated for culture. The enzymatic digestion process involves first using collagenase IV for the first step, with a digestion time of 18-22 min, followed by a second step using a complex enzyme consisting of collagenase IV and trypsin for the second step, with a digestion time of 8-12 min. The basal culture medium is DMEM medium without NaHCO3. 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, specifically 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: In the first step of enzyme digestion, the concentration of collagenase IV is 0.8~1.5 mg / mL. In the second step of enzyme digestion, the complex enzyme consists of 1 mg / mL collagenase IV and 0.25% trypsin in a 1:1 volume ratio.

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 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℃.

5. The method for isolating and culturing granulosa cells from the ovaries of the Chinese soft-shelled turtle as described in claim 4, 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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