Isolated culture method of functional Chinese softshell turtle ovarian granular cells

Abstract

The invention relates to an isolated culture method of functional Chinese softshell turtle ovarian granular cells. The isolated culture method comprises the following steps: S1, collecting ovarian tissues: collecting Chinese softshell turtle ovarian tissues; s2, separating and collecting a granular cell layer; s3, digesting and collecting granular cells; s4, primary culture of granulosa cells: transferring the collected cells into a cell culture bottle containing 4 ml of a complete culture medium for primary culture, wherein the complete culture medium comprises the following main components: a DMEM culture medium, 15% of fetal calf serum, 1% of penicillin streptomycin and 1% of an insulin-transferrin-selenium additive; and S5, subculturing the granular cells. The isolated culture method and the culture medium system provided by the invention are simpler and more time-saving and labor-saving; according to the method, the aromatase CYP19A1 can be expressed normally, the characteristics of the granular cells are kept, the aromatase CYP19A1 can be expressed normally, it is indicated that the in-vitro cultured cells can keep the functionality of the cells, and the in-vitro proliferation capacity of the granular cells is improved to a great extent.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal cell culture technology, and in particular to a method for isolating and culturing granulosa cells of the ovary of the Chinese soft-shelled turtle. Background Technology

[0002] The Chinese soft-shelled turtle (Pelodiscus sinensis), commonly known as the turtle, has delicious, meaty flesh. Its head, carapace, shell, and internal organs are all used in traditional Chinese medicine, making it one of my country's prized aquatic farm species. Granulosa cells, as an important component of follicles, play a crucial role in regulating hormone secretion during follicle development. Therefore, establishing a granulosa cell culture model is essential for studying Chinese soft-shelled turtle follicle development and improving its egg-laying efficiency.

[0003] Current techniques for isolating and culturing granulocytes mainly focus on humans and chickens, with no reports on the Chinese soft-shelled turtle (Typhoid Fern). Chicken granulocyte culture medium is unsuitable for culturing Chinese soft-shelled turtle granulocytes, resulting in a high mortality rate. Establishing a method for isolating and culturing Chinese soft-shelled turtle granulocytes in vitro could alleviate the time-consuming process of primary cell isolation, reduce animal mortality, and contribute to improving the reproductive efficiency of the Chinese soft-shelled turtle. Summary of the Invention

[0004] This invention provides a method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] A method for isolating and culturing functional ovarian granulosa cells of the Chinese soft-shelled turtle includes the following steps:

[0007] S1. Ovarian tissue collection: Ovarian tissue from Chinese soft-shelled turtles was collected.

[0008] S2. Separation and collection of granular cell layer;

[0009] S3, digestion and collection of granulocytes;

[0010] S4. Primary culture of granulocytes: The collected cells were transferred to cell culture flasks containing 4 ml of complete culture medium for primary culture. The main components of the complete culture medium were: DMEM medium, 15% fetal bovine serum, 1% penicillin-streptomycin, and 1% insulin-transferrin-selenium additive.

[0011] S5. Granulosa cell passage culture.

[0012] Preferably, in step S1, ovarian tissue from a 1-year-old Chinese soft-shelled turtle is taken.

[0013] Preferably, in step S1, the specific steps are: cleaning the tissue surface with PBS and placing it in a clean, sterile culture dish containing PBS.

[0014] Preferably, in step S2, the specific steps are as follows: separate the Chinese soft-shelled turtle follicles with a diameter of 5-8 mm, puncture the follicle wall with ophthalmic forceps, and clean the yolk material inside the follicle; after cleaning, gently turn the follicle membrane over and gently shake to separate the granulosa cell layer.

[0015] Preferably, in step S3, the specific steps are as follows: the separated granular cells are collected in a sterile milliliter tube, digested with 2 mg / ml type II collagenase at 30° for 8 minutes, and then fetal bovine serum is added to terminate the digestion; the digested cells are filtered through a 200-mesh cell sieve and placed in a 15 ml centrifuge tube, centrifuged at 800 x g for 10 minutes, the supernatant is discarded, and the cells are resuspended in complete culture medium to obtain a cell suspension.

[0016] Preferably, in step S4, the conditions for primary culture of granulocytes are: placed in a 5% CO2, 30°C incubator; the medium is changed every 24-48 hours.

[0017] Preferably, in step S5, the specific steps are as follows: after the primary cells have proliferated to a cell density of 90%, the culture medium is discarded, 1 ml of PBS is added to wash the adherent cells twice, 1 ml of 0.25% trypsin is added and digested at 37°C for 5 minutes until the cells become round, fetal bovine serum is added to stop the digestion, the mixed liquid is pipetted and mixed well, and then all of it is transferred to a 15 ml tube, centrifuged at 800 x g for 10 minutes and the supernatant is discarded, the cells are resuspended in complete culture medium to obtain a cell suspension, and then all of it is transferred to a cell culture flask for passage culture.

[0018] Preferably, in step S5, before transferring the cells to the cell culture flask, 50 μg / ml rat tail collagen I is evenly coated on the cell culture flask at a concentration of 5 μg / cm2, and after standing at room temperature for 1 hour, the cells are washed clean with PBS.

[0019] Preferably, in step S4, 10 ng / ml of LIF and FGF are added to the complete culture medium.

[0020] Compared with existing technologies, the functional method for isolating and culturing Chinese soft-shelled turtle ovarian granulosa cells of this invention provides a simpler culture medium system for culturing these cells, eliminating the need for pre-coating with rat tail collagen and saving time and effort. Early studies have shown that granulosa cells gradually luteinize and undergo apoptosis after 6-24 hours of in vitro culture. However, the method of this invention allows for passage culture of granulosa cells up to the fifth generation, and some cells express the granulosa cell-specific protein CYP19A1, indicating that the in vitro cultured cells can maintain their functionality. This significantly improves the in vitro proliferation efficiency of cells, providing an in vitro technology platform for research on the reproductive development of Chinese soft-shelled turtles and related fields. Establishing a functional method for isolating and culturing Chinese soft-shelled turtle granulosa cells can alleviate the difficulty of time-consuming primary cell isolation, reduce animal mortality, and contribute to improving the reproductive efficiency of Chinese soft-shelled turtles. Attached Figure Description

[0021] Figure 1 This is a three-dimensional microscopic photograph of adherent granulosa cells in Example 1 of the functional Chinese soft-shelled turtle ovarian granulosa cell isolation and culture method of the present invention; the scale bar in the figure is 50 μm.

[0022] Figure 2 This invention provides RT-qPCR verification of genes specifically expressed or highly expressed in granulosa cells in Example 1 of the method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells.

[0023] Figure 3 The image shows the immunofluorescence staining results of FOXL2 and CYP19A1, specifically expressed proteins of granulosa cells, in Example 1 of the functional Chinese soft-shelled turtle ovarian granulosa cell isolation and culture method of the present invention; the scale bar in the figure is 50 μm.

[0024] Figure 4 This is a three-dimensional microscopic photograph of adherent granulosa cells under a microscope in Example 2 of the functional Chinese soft-shelled turtle ovarian granulosa cell isolation and culture method of the present invention; the scale bar in the figure is 100 μm.

[0025] Figure 5 This invention provides a comparison of the survival rate of Chinese soft-shelled turtle granulosa cells in Example 3 and Example 1 regarding the isolation and culture method of functional Chinese soft-shelled turtle ovarian granulosa cells. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0027] Example 1:

[0028] A method for isolating and culturing functional ovarian granulosa cells of the Chinese soft-shelled turtle, specifically including the following steps:

[0029] S1. Ovarian tissue collection: Ovarian tissue from 1-year-old Chinese soft-shelled turtles was collected, and the tissue surface was cleaned with PBS to remove blood and dirt. The tissue was then placed in a clean, sterile culture dish containing PBS.

[0030] S2. Separation and collection of granulosa cell layer: Chinese soft-shelled turtle follicles with a diameter of 5-8 mm are separated individually. The follicle wall is punctured with ophthalmic forceps, and the yolk material inside the follicle is cleaned. After cleaning, the follicle membrane is gently turned over and the granulosa cell layer is gently shaken to separate it.

[0031] S3. Digestion and collection of granulocytes: Collect the isolated granulocytes in sterile mL tubes, digest them with 2 mg / mL type II collagenase at 30°C for 8 minutes, and then add fetal bovine serum to stop the digestion; after digestion, filter the cells through a 200-mesh cell sieve and place them in a 15 mL centrifuge tube. Centrifuge at 800 x g for 10 minutes, discard the supernatant, and resuspend the cells in complete culture medium to obtain a cell suspension.

[0032] S4. Primary culture of granulocytes: The collected cells were transferred to cell culture flasks containing 4 ml of complete culture medium and placed in a 5% CO2, 30°C incubator for primary cell culture; the cell adhesion status was observed and photographed under a microscope; the medium was changed every 24-48 hours; the main components of the complete culture medium were: DMEM medium, 15% fetal bovine serum, 1% penicillin-streptomycin, and 1% insulin-transferrin-selenium additive.

[0033] S5. Granulosa cell passage culture: After the primary cells have proliferated to a cell density of 90%, discard the culture medium, add 1 ml PBS to wash the adherent cells twice, add 1 ml 0.25% trypsin and digest at 37°C for 5 minutes until the cells become rounded, add fetal bovine serum to stop the digestion, mix the mixture by pipetting and transfer it to a 15 ml tube, centrifuge at 800xg for 10 minutes and discard the supernatant, resuspend in complete culture medium to obtain a cell suspension, and then transfer it to a cell culture flask for passage culture.

[0034] Granulosa cells were collected after third-generation subculture, RNA was extracted and reverse transcribed into cDNA, and the expression of granulosa cell-specific genes such as WNT4, FOXL2, and CYP19A1 was verified using RT-qPCR. Figure 2 As shown, the results indicate that the expression levels of WNT4, FOXL2, and CYP19A1 in granulosa cells are significantly higher than those in other follicular tissues, demonstrating the accuracy of granulosa cell isolation.

[0035] Cells were collected after third-generation passage for immunofluorescence staining of granulocyte-specific proteins FOXL2 and CYP19A1, such as... Figure 3As shown, the results indicate that the granulocytes cultured in vitro specifically express the protein FOXL2, which overlaps with the nucleic acid dye DAPI, indicating that the granulocytes cultured in vitro have high purity; some granulocytes can express CYP19A1, which is expressed in the cytoplasm, indicating that the granulocytes cultured and passaged in vitro maintain granulocyte specificity.

[0036] Example 2:

[0037] Same as Example 1, except that 10 ng / ml of LIF and FGF were added to the complete culture medium. Cell morphology observation of the granular cells obtained after three passages showed that the cell state was consistent with that of Example 1. Figure 4 As shown, the cells adhere well to the wall, and cells undergoing division and proliferation are visible.

[0038] Example 3:

[0039] Same as Example 1, except that before transferring the cells to the cell culture flask, 50 μg / ml rat tail collagen I was used at a concentration of 5 μg / cm. 2 Spread the mixture evenly onto a cell culture flask, incubate at room temperature for 1 hour, then wash thoroughly with PBS. Passage to the third generation and collect cells for CCK-8 cell proliferation rate assays. Figure 5 As shown, there was no significant difference in the cell proliferation rate between Example 1 and Example 3.

[0040] This invention provides a simpler culture medium system for culturing Chinese soft-shelled turtle ovarian granulosa cells, eliminating the need for pre-coating with rat tail collagen and saving time and effort. Early studies showed that granulosa cells gradually luteinize and undergo apoptosis after 6-24 hours of in vitro culture. However, the method of this invention allows for passage culture of granulosa cells up to the fifth generation, and some cells express the granulosa cell-specific protein CYP19A1, indicating that the in vitro cultured cells maintain their functionality. This significantly improves the in vitro proliferation efficiency of cells, providing an in vitro technology platform for research on the reproductive development of Chinese soft-shelled turtles and related fields. Establishing a functional method for isolating and culturing Chinese soft-shelled turtle granulosa cells can alleviate the difficulty of time-consuming primary cell isolation, reduce animal mortality, and contribute to improving the reproductive efficiency of Chinese soft-shelled turtles.

[0041] Finally, it should be noted that the above embodiments are only illustrative of the technical solutions of the present invention, and not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for isolating and culturing functional granulosa cells from the ovaries of the Chinese soft-shelled turtle, characterized in that, Includes the following steps: S1. Ovarian tissue collection: Ovarian tissue from Chinese soft-shelled turtles was collected. S2. Separation and collection of granular cell layer; S3, digestion and collection of granulocytes; S4. Primary culture of granulocytes: The collected cells were transferred to cell culture flasks containing 4 ml of complete culture medium for primary culture. The main components of the complete culture medium were: DMEM medium, 15% fetal bovine serum, 1% penicillin-streptomycin, and 1% insulin-transferrin-selenium additive. S5. Granulosa cell passage culture.

2. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 1, characterized in that, In step S1, the ovarian tissue of a 1-year-old Chinese soft-shelled turtle is used.

3. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 1 or 2, characterized in that, In step S1, the specific steps are as follows: clean the tissue surface with PBS and place it in a clean, sterile culture dish containing PBS.

4. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 2, characterized in that, In step S2, the specific steps are as follows: separate the Chinese softshell turtle follicles with a diameter of 5-8 mm, puncture the follicle wall with ophthalmic forceps, and clean the yolk material inside the follicle; after cleaning, gently turn the follicle membrane over and gently shake to separate the granulosa cell layer.

5. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 1, characterized in that, In step S3, the specific steps are as follows: the separated granular cells are collected in a sterile milliliter tube, digested with 2 mg / ml type II collagenase at 30°C for 8 minutes, and then fetal bovine serum is added to terminate the digestion; the digested cells are filtered through a 200-mesh cell sieve and placed in a 15 ml centrifuge tube, centrifuged at 800 xg for 10 minutes, the supernatant is discarded, and the cells are resuspended in complete culture medium to obtain a cell suspension.

6. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 1, characterized in that, In step S4, the conditions for primary culture of granulocytes are: placed in a 5% CO2, 30℃ incubator; the medium is changed every 24-48 hours.

7. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 1, characterized in that, In step S5, the specific steps are as follows: after the primary cells have proliferated to a cell density of 90%, the culture medium is discarded, 1 ml of PBS is added to wash the adherent cells twice, 1 ml of 0.25% trypsin is added and digested at 37°C for 5 minutes until the cells become round, fetal bovine serum is added to stop the digestion, the mixed liquid is pipetted and mixed well, and then all of it is transferred to a 15 ml tube, centrifuged at 800xg for 10 minutes and the supernatant is discarded, the cells are resuspended in complete culture medium to obtain a cell suspension, and then all of it is transferred to a cell culture flask for passage culture.

8. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 7, characterized in that, In step S5, before transferring the cells to the cell culture flask, 50 μg / ml rat tail collagen I is evenly coated on the cell culture flask at a concentration of 5 μg / cm2. After standing at room temperature for 1 hour, the cells are washed clean with PBS.

9. The method for isolating and culturing functional Chinese soft-shelled turtle ovarian granulosa cells according to claim 1, characterized in that, In step S4, 10 ng / ml of LIF and FGF are added to the complete culture medium.

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