Method for culturing primary spermatogonial stem cells of Tibetan sheep

Abstract

The invention provides a method for culturing primary spermatogonial stem cells of Tibetan sheep, and belongs to the technical field of cell separation and culture. The culture method provided by the invention comprises the following steps: disinfecting and cleaning testis tissues of Tibetan sheep lambs; shearing tissue blocks, and digesting and cleaning to obtain a single-cell suspension of the seminiferous tubule; purifying the single-cell suspension, and culturing without a feeding layer or with the feeding layer; the culture without the feeding layer refers to culture in a laminin-coated culture container by using a feeding-layer-free MEM alpha culture medium or a StemProTM-34SFM complete culture medium; culture with a feeding layer refers to culture in a mouse fibroblast line feeding layer or a Tibetan sheep primary testicular sertoli feeding layer, and a feeding layer MEMS alpha culture medium is added. According to the culture method provided by the invention, the Tibetan sheep spermatogonial stem cells with higher purity can be obtained through separation, the subsequent proliferation of the spermatogonial stem cells can be remarkably promoted, and the apoptosis of the spermatogonial stem cells can be inhibited.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation and culture, and in particular to a method for culturing primary spermatogonial stem cells of Tibetan sheep. Background Art

[0002] Spermatogonial stem cells (SSCs) are a type of reproductive stem cell in the testes of male animals that can proliferate and differentiate to produce sperm and pass on genetic information to the next generation. In theory, SSCs can proliferate in large numbers in vitro and are an ideal cell source in the fields of transgenic animal production, germplasm resource protection, and reproductive medicine development. At present, the in vitro culture technology of spermatogonial stem cells is relatively mature in model animals such as mice and rats, but progress is slow in large animals. However, to date, there is still no long-term culture system that can support the proliferation and maintenance of SSCs in large animals such as livestock, and the cultured SSCs cannot reconstruct donor-derived spermatogenesis in the testes of the same species with immune deficiency. The establishment of a long-term in vitro culture system for large animals is not only helpful for studying the biological functions of SSCs, but also crucial for the application of SSCs (such as genetic modification, assisted reproduction, etc.). Therefore, in-depth research on the long-term in vitro culture conditions of SSCs in large animals such as livestock has laid the foundation for the subsequent application of SSCs in practice. Summary of the invention

[0003] The purpose of the present invention is to provide a method for culturing primary spermatogonial stem cells of Tibetan sheep, so as to lay a foundation for the subsequent application of SSCs in practice.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for culturing primary spermatogonial stem cells of Tibetan sheep, comprising the following steps:

[0006] (1) obtaining testicular tissue of Tibetan sheep lambs, disinfecting and cleaning them, and obtaining tissue blocks;

[0007] (2) mincing the tissue blocks, digesting, washing, and resuspending to obtain a single cell suspension of the seminiferous tubules;

[0008] (3) purifying the single cell suspension to obtain Tibetan sheep primary spermatogonial stem cells;

[0009] (4) culturing the Tibetan sheep primary spermatogonial stem cells without or with a feeder layer;

[0010] The feeder-free culture is carried out using feeder-free MEMα medium or StemPro TM -34SFM complete medium was cultured in laminin-coated culture vessels;

[0011] The feeder layer culture is cultured in a mouse fibroblast cell line feeder layer or in a Tibetan sheep primary testicular supporting cell feeder layer, and a feeder layer MEMα culture medium is added.

[0012] Preferably, the preparation method of the primary testicular support cell feeder layer of Tibetan sheep is as follows: fresh testicular tissue of the Tibetan sheep lamb in step (1) is collected, processed into sterile tissue blocks, digested with collagenase IV and trypsin in sequence, and after the digestion is terminated, the digestion solution is sieved step by step to collect cells; after the cells are resuspended, they are separated using Percoll cell separation solution, and the suspended cell solution with 11% to 19% and 27% to 35% Percoll gradients is collected and purified to obtain purified primary testicular support cells of Tibetan sheep; then the primary testicular support cells of Tibetan sheep are treated with mitomycin C, and the treated primary testicular support cells of Tibetan sheep are prepared into a cell suspension, and the cell suspension is cultured into a monolayer of cells in a culture bottle pre-coated with 0.1% gelatin to obtain a primary testicular support cell feeder layer of Tibetan sheep.

[0013] Preferably, the sieves through which the digestion fluid is screened step by step are 80-100 mesh, 170-200 mesh, and 270-300 mesh, respectively; and the gradient concentrations of the Percoll cell separation fluid are 10-12%, 18-20%, 26-28%, 34-36%, and 42-44%.

[0014] Preferably, the feeder-free MEMα culture medium is prepared in a 50 mL system and the following components are added: 48-49 mL MEMα culture medium, 0.4-0.6 mL FBS, 0.4-0.6 mL penicillin-streptomycin, 95-105 μL 18-22 ng / mL GDNF, 45-55 μL 9-11 ng / mL bFGF, 45-55 μL 900-1100 U / mL LIF and 45-55 μL 9-11 ng / mL SDF-1.

[0015] Preferably, the StemPro TM -34SFM medium was prepared according to the 50mL system and the following components were added: 44-45mL StemPro TM-34SFM medium, 0.4~0.6mL FBS, 0.4~0.6mL penicillin-streptomycin, 0.4~0.6mL 1% non-essential amino acids, 45~55μL 10μg / mL insulin, 45~55μL 10μg / mL transferrin, 0.4~0.6mL 1% β-mercaptoethanol, 0.4~0.6mL 1% sodium pyruvate, 45~55μL 19~21μg / mL putrescine, 0.4~0.6mL 1% L-glutamine, 0.4~0.6mL 1% vitamins, 0.4~0.6mL 9~11mM Hepes buffer, 0.8~1.2mL 2% StemPro neural supplement, 90~110μL 19~21ng / mL GDNF, 45~55μL 9~11ng / mLbFGF, 45~55μL 1000U / mL LIF and 45~55μL 9~11ng / mL SDF-1.

[0016] Preferably, the feeder layer MEMα culture medium is prepared according to a 50 mL system, and the following components are added: 48-49 mL MEMα culture medium, 0.4-0.6 mL FBS, 0.4-0.6 mL penicillin-streptomycin, 90-110 μL 19-21 ng / mL GDNF, 48-52 μL 9-11 ng / mL bFGF, 48-52 μL 900-1100 U / mL LIF and 48-52 μL 9-11 ng / mL SDF-1.

[0017] The present invention provides a method for culturing Tibetan sheep primary spermatogonial stem cells, which can separate Tibetan sheep spermatogonial stem cells with higher purity, and can significantly promote the subsequent proliferation of spermatogonial stem cells and inhibit their apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the picture of the 10× mouse fibroblast cell line in Example 1;

[0019] Figure 2 This is the isolation and identification of Tibetan sheep supporting cells in Example 1; A: primary Tibetan sheep supporting cells (20×), the oval ones are immature SCs, and the spindle ones are mature SCs; B: Oil Red O staining identification results; C: Immunofluorescence staining identification results.

[0020] Figure 3 The results of the isolation and identification of Tibetan sheep spermatogonial stem cells in Example 1; A: spermatogonial stem cells just isolated (4×); B: spermatogonial stem cells after purification; C: immunofluorescence staining identification results.

[0021] Figure 4 This is a representative image of the proliferation of spermatogonial stem cell feeder layer system in Example 1 (10×);

[0022] Figure 5 The effects of different feeder layers on the proliferation of SSCs in Example 1; A: The changes in the growth fold of spermatogonial stem cells in different feeder layers; B: The CCK8 method was used to detect the effects of different feeder layers on the proliferation of SSCs; C: The EdU labeling method was used to detect the effects of different feeder layers on the proliferation of SSCs; D: The immunofluorescence method was used to detect the effects of different feeder layers on the proliferation of SSCs;

[0023] Figure 6 The effect of different feeder layers on the self-renewal and differentiation gene expression of spermatogonial stem cells in Example 1;

[0024] Figure 7 This is a representative image of the proliferation of spermatogonial stem cells in the feeder-free system of Example 1 (10×);

[0025] Figure 8 The effects of different feeder layers on the proliferation of SSCs in Example 1; A: The changes in the growth fold of spermatogonial stem cells in different feeder layers; B: The CCK8 method was used to detect the effects of different feeder layers on the proliferation of SSCs; C: The EdU labeling method was used to detect the effects of different feeder layers on the proliferation of SSCs; D: The immunofluorescence method was used to detect the effects of different feeder layers on the proliferation of SSCs;

[0026] Fig. 9 This is the effect of different culture media on the expression of spermatogonial stem cell self-renewal and differentiation genes in Example 1. DETAILED DESCRIPTION

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Test materials

[0029] 2-month-old male Tibetan sheep lamb

[0030] Experimental preparation

[0031] Sterile scissors, forceps, clean bench, 75% ethanol, penicillin-streptomycin (gibco, 15140163), PBS buffer (Solar, P1020), HBSS buffer (gibco, 14175095), T25 culture flask (Corning, CLS430639), 6-well plate (Costar, 3516), 96-well plate (Corning, 3799), 0.25% trypsin (gibco, 15050065), collagenase type IV (sigma, C5138), DNaseⅠ enzyme (Sigma, 10104159001), 1% gelatin (Solar, G0040), Percoll cell separation solution (Sigma, P1644), MEMα medium (gibco, 41061029), StemPro TM -34SFM (gibco, 10639011), fetal bovine serum (gibco, 10099-141C), laminin (ZETA, Z10255), non-essential amino acids (Solebol, N1250), insulin (Sigma, Y0001717), transferrin (Sigma, T1283), β-mercaptoethanol (Macklin, M828395), sodium pyruvate (Solebol, SP0100), putrescine (Sigma, P5780), L-glutamine ( Solebao, G0200), MEM vitamin solution (gibco, 11120052), Hepes buffer (Sigma, H0887-100ML), StemPro neural additive (gibco, A1000601), GDNF (Peprotech, 450-51-10UG), bFGF (Sigma, GF446), LIF (Sigma, ESG1106), SDF-1 (Sigma, SRP3276), Rabbit Anti-Plzf antibody (abcam, ab-104854), Rabbit Anti-UCHL1+3antibody (Boaosen, bs-11677R), GoatAnti-Rabbit IgG H&Lantibody (Boaosen, bs-0295G-Cy3), RabbitAnti-GATA4 (Boaosen, bs-23982R), alkaline phosphatase staining kit (Solebio, G1480), and mitomycin C (Aladdin, D419552).

[0032] Reagent preparation

[0033] ① Type IV collagenase: Weigh 5 mg of type IV collagenase and dissolve it in 20 mL of PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare a 0.25 mg / mL cell digestion solution;

[0034] ②DNaseⅠ enzyme: weigh 175 mg DNaseⅠ enzyme and dissolve it in 25 mL PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare 7 mg / mL cell digestion solution;

[0035] ③1% gelatin (Solabo, G0040): Pipette 1 mL of 1% gelatin solution and add it to 9 mL of PBS without calcium and magnesium to dilute it into 0.1% gelatin solution;

[0036] ④20ng / mL GDNF: Take 1mL of deionized water and add it to 10μg of GDNF freeze-dried powder. After fully dissolving, prepare a 10μg / mL GDNF working solution;

[0037] ⑤10ng / mL bFGF: Take 1mL of PBS without calcium and magnesium, add it to 10μg bFGF lyophilized powder, and prepare 10μg / mL bFGF working solution after fully dissolving;

[0038] ⑥10ng / mL SDF-1: Take 1mL of deionized water and add it to 10μg of SDF-1 lyophilized powder. After fully dissolving, prepare a 10μg / mL SDF-1 working solution;

[0039] ⑦10μg / mL insulin: Take 1mL of deionized water and add it to 10mg of insulin. After fully dissolving, prepare 10mg / mL insulin working solution;

[0040] ⑧10 μg / mL transferrin: Take 1 mL of deionized water and add it to 10 mg of transferrin. After fully dissolving, prepare a 10 mg / mL transferrin working solution;

[0041] ⑨20μg / mL putrescine: Take 1mL of deionized water and add it to 20mg of putrescine. After fully dissolving, prepare a 20mg / mL putrescine working solution.

[0042] Preparation of complete culture medium

[0043] a. Feeder layer MEMα medium (50 mL): 48.75 mL MEMα medium, 0.5 mL FBS, 0.5 mL penicillin-streptomycin, 100 μL 20 ng / mL GDNF, 50 μL 10 ng / mL bFGF, 50 μL 1000 U / mL LIF and 50 μL 10 ng / mL SDF-1;

[0044] b. Feeder-free MEMα medium (50 mL): 44.6 mL MEMα medium, 0.5 mL FBS, 0.5 mL penicillin-streptomycin, 0.5 mL 1% non-essential amino acids, 50 μL 10 μg / mL insulin, 50 μL 10 μg / mL transferrin, 0.5 mL 1% β-mercaptoethanol, 0.5 mL 1% sodium pyruvate, 50 μL 20 μg / mL putrescine, 0.5 mL 1% L-glutamine, 0.5 mL 1% vitamins, 0.5 mL 10 mM Hepes buffer, 1 mL 2% StemPro neural supplement, 100 μL 20 ng / mL GDNF, 50 μL 10 ng / mL bFGF, 50 μL 1000 U / mL LIF and 50 μL 10 ng / mL SDF-1;

[0045] c. StemPro without feeder layer TM -34SFM medium (50mL): 44.6mL StemPro TM -34SFM medium, 0.5mL FBS, 0.5mL penicillin-streptomycin, 0.5mL 1% non-essential amino acids, 50μL 10μg / mL insulin, 50μL 10μg / mL transferrin, 0.5mL 1% β-mercaptoethanol, 0.5mL 1% sodium pyruvate, 50μL 20μg / mL putrescine, 0.5mL 1% L-glutamine, 0.5mL 1% vitamins, 0.5mL 10mM Hepes buffer, 1mL 2% StemPro neural supplement, 100μL 20ng / mL GDNF, 50μL 10ng / mL bFGF, 50μL 1000U / mL LIF and 50μL 10ng / mL SDF-1.

[0046] Example 1

[0047] 1.1 Isolation, purification and culture of primary spermatogonial stem cells

[0048] (1) Take lamb testicular tissue and sterilize it in 75% ethanol for 1 min;

[0049] (2) Wash three times in PBS containing 1% PS in a clean bench;

[0050] (3) Use sterile scissors to cut off the outer albuginea and epididymis of the testis, avoid the external blood vessels of the testis, cut the testis in half, take about 200 mg of tissue blocks from the middle of each testis, transfer the tissue blocks to a culture dish containing 2 mL of HBSS, and cut them into small pieces of 0.5 × 0.5 cm with scissors. Add 20 ml of 0.25 mg / ml type IV collagenase and 1 ml of 7 mg / ml DNase I to a 50 mL centrifuge tube, transfer the above-cut tissue into the tube, digest at 37°C for 7 min, centrifuge at 300 g for 1 min at 4°C, discard the supernatant, add 3 ml of HBSS to the precipitate, transfer the digestion solution to a 15 ml centrifuge tube, and place the tube in an ice box for 2 min to allow it to settle naturally, then discard the supernatant. Wash five times according to this step. For the last wash, centrifuge at 300 g for 1 min at 4°C and discard the supernatant. Add 8ml 0.25% trypsin and 2ml DNaseⅠ, mix well and place in a 37℃ water bath for digestion for 25min, shaking by hand every 5min. Add 1.5ml DMEM / F-12 culture medium and 0.5ml DNaseⅠ to the above tube, centrifuge at 600g 4℃ for 7min and discard the supernatant. Add 5ml DNaseⅠ and 2ml HBSS, mix well with a pipette tip and pour into a 100μm cell sieve for filtration, centrifuge the above cell suspension at 600g 4℃ for 7min and discard the supernatant, then wash the precipitate twice with 5ml HBSS to obtain a single cell suspension of seminiferous tubules;

[0051] (4) Add 2 ml of percoll working solution to a 15 mL centrifuge tube, then add 5 ml of single cell suspension of seminiferous tubules, mix well, centrifuge at 600 g, 4°C for 8 min, and discard the supernatant. Add 3 ml of HBSS to the tube for washing, centrifuge at 600 g, 4°C for 7 min, and discard the supernatant. Use the differential adhesion method to enrich spermatogonial stem cells SSCs. The specific steps are as follows: Take a T25 culture flask, add 2 ml of 0.1% gelatin aqueous solution to coat the bottom, place it in a 37°C incubator for 30 min, discard the solution, and rinse with HBSS. Resuspend the cells in the centrifuge tube in DMEM / F-12 medium containing 5% FBS, inoculate the cell suspension into the culture flask before the culture flask dries, and place it in a 37°C 5% CO 2 Culture in an incubator. Culture for 2 hours, which is the first differential adhesion. Collect the non-adherent cell suspension and centrifuge, resuspend in DMEM / F-12 medium containing 5% FBS, transfer to a new culture flask coated with gelatin in advance and continue to culture for 4 hours, which is the second differential adhesion. Centrifuge the non-adherent cell suspension again, transfer to a new culture flask coated with gelatin in advance and continue to culture for 24 hours, which is the third differential adhesion. Collect the non-adherent cell suspension, centrifuge at 1200rpm for 5 minutes, and discard the supernatant to obtain SSCs with a purity of more than 80%.

[0052] (5) Preparation of feeder layer cells:

[0053] ① Mouse fibroblast cell line (MEF)

[0054] Mouse fibroblast cell line was purchased from Beina Biotechnology (BNCC328309). MEF cell line was revived by conventional cell culture method. 0.5mg / ml 100μL mitomycin C was added to T25 culture flask and incubated in 37℃ incubator for 2h. After incubation, the culture medium was aspirated and rinsed three times with HBSS. 0.25% trypsin was added to digest the cells and incubated at 37℃ for 3min. 2ml culture medium was added to each flask to terminate the digestion. The cell suspension was aspirated into a centrifuge tube and centrifuged at 800rpm for 5min. The supernatant was discarded and the cells were resuspended in the culture medium. The cell suspension was cultured in a culture flask pre-coated with 0.1% gelatin. Within 48h, the cells formed a monolayer at the bottom of the culture flask and were used as a feeder layer for spermatogonial stem cells.

[0055] ②Tibetan sheep primary testicular supporting cells (SCs)

[0056] Fresh testicular tissue from the same Tibetan sheep lamb was collected, the surface capsule was cut open on a clean bench, the sterile part was taken out, and it was washed three times with PBS containing 1% double antibody. Then 5 times the volume of collagenase IV was added, placed in a 37°C water bath for digestion for 1h, shaken once every 10min, centrifuged at 800rpm for 5min, and the supernatant was discarded. Then 0.25% trypsin was added, placed in a 37°C water bath for digestion for 15min, shaken once every 5min, and finally an equal volume of complete medium was added to terminate the digestion. The cells were sieved step by step with 100 mesh, 200 mesh, and 300 mesh cell sieves, respectively, and the liquid under the sieve was centrifuged at 1000rmp / min for 5min, and the supernatant was discarded. The Percoll cell separation solution was prepared into solutions with volume fractions of 11%, 19%, 27%, 35%, and 43%, respectively, using PBS containing 1% double antibody. Resuspend the cells with 1mL PBS, take the prepared Percoll cell separation solution for each grade, add it to a 15mL centrifuge tube according to the density from high to low, gently add the cell suspension to be separated to the top layer of the gradient, and centrifuge at 1400r / min for 20min. Transfer the suspension of 11%-19% and 27%-35% Percoll gradient to a new 15mL centrifuge tube, wash twice with PBS containing 1% double antibody, and add an appropriate amount of complete culture medium. For further purification, use a hypotonic solution of 0.05% trypsin to digest the cells during cell passage to remove the interstitial cells that were not completely removed in the previous step. After the cells grow for 6h, replace the culture medium to remove the spermatogenic cells that are not attached to the wall. Repeat the above operation 3 times to obtain SCs with higher purity, treat the cells with mitomycin C, and make a feeder layer. The specific operation steps are the same as those of the MEF cell line.

[0057] (6) Culture of feeder layer system: Remove the culture medium in the feeder layer and wash it with HBSS 4 times. The purified spermatogonial stem cells were repeatedly blown and collected by centrifugation, resuspended with 2 mL of prepared feeder layer MEMα medium and inoculated into two feeder layers of mouse embryonic fibroblast cell line and Tibetan sheep testicular supporting cells at a ratio of 1:1, and then 3 mL of prepared feeder layer MEMα medium was added. The cells were placed in an incubator at 37°C and 5% CO 2 To cultivate.

[0058] (7) Laminin-coated culture flask: Take 1 tube of laminin (125 μL, 125 μg) and thaw it at room temperature. Prepare a 15 mL centrifuge tube, take 2 mL of DPBS, add the thawed laminin into DPBS, and gently mix the diluted laminin. Use the diluted laminin working solution to coat the T25 culture flask. Gently shake the culture flask to spread the diluted laminin working solution evenly on the bottom surface of the flask. Let it stand at room temperature for at least 1 hour before use. When using, tilt the culture flask and use the gun tip to absorb the coating solution to ensure that there are no scratches on the bottom surface of the coated culture flask. The culture flask is used for the subsequent culture of spermatogonial stem cells without a feeder layer system.

[0059] (8) Cultivation without feeder layer system: The purified spermatogonial stem cells were repeatedly blown and collected by centrifugation, and then the cells were counted and 3×10 5 5 ml feeder-free MEMα and StemPro TM Resuspend spermatogonial stem cells in -34SFM complete medium and inoculate them into culture flasks coated with laminin. Place the cells in an incubator at 37°C and 5% CO 2 To cultivate.

[0060] 1.2 Experimental methods

[0061] 1.2.1 Alkaline phosphatase staining steps

[0062] (1) Digest the spermatogonial stem cells cultured in 1.1 and prepare a cell suspension, centrifuge at 1200 rpm for 5 min, and discard the supernatant;

[0063] (2) Resuspend in 1 mL PBS and inoculate into a six-well plate, and bake in a 60°C oven for 30 min;

[0064] (3) Add ALP fixative for 3 min and wash with distilled water;

[0065] (4) Add ALP incubation solution, place in a wet box, incubate for 15 min away from light, and wash with distilled water;

[0066] (5) Add nuclear fast red staining solution or methyl green staining solution for 4 minutes, wash with PBS, and examine under a microscope.

[0067] 1.2.2 Immunofluorescence staining

[0068] (1) Use a PAPPEN immunohistochemistry pen to draw circles on an anti-shedding slide, drop spermatogonial stem cell suspension into each circle, and bake in a 60°C oven for 30 min;

[0069] (2) Add 4% paraformaldehyde for 15 min;

[0070] (3) Wash with PBS three times, 5 min each time;

[0071] (4) Add 0.3% TritonX-100 for permeabilization for 10 min;

[0072] (5) Wash with PBS three times, 5 min each time;

[0073] (6) Add blocking solution and block at room temperature for 60 min;

[0074] (7) Add primary antibodies GATA4 (1:200), UCHL1 (1:200), and PLZF (1:200) and incubate at 4°C overnight;

[0075] (8) Wash with PBST three times, 5 min each time;

[0076] (9) Add goat anti-rabbit secondary antibody (1:200) and incubate at room temperature in the dark for 1 h;

[0077] (10) Wash with PBST three times, 5 min each time;

[0078] (11) DAPI solution was added to stain the cell nuclei and incubated at room temperature in the dark for 10 min;

[0079] (12) Wash with PBST three times, 5 min each time;

[0080] (13) Observe the cells under a confocal microscope.

[0081] 1.2.3CCK-8 proliferation assay

[0082] (1) Cells were seeded into 96-well plates, 100 μl of culture medium was added to each well, and the plates were incubated at 37°C and 5% CO. 2 Culture in an incubator for 48 h;

[0083] (2) CCK-8 reagent (10 μL) was added to the culture medium in each well;

[0084] (3) After incubation in the incubator for 2 h, the optical density (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader, with 6 replicates per group.

[0085] 1.2.4 EdU proliferation assay

[0086] (1) Take cells in logarithmic growth phase and add 1×10 4 The cells were seeded in 96-well plates, with three replicates for each treatment, and 100 μL of complete medium was added to each well and the cells were cultured for 24 h before the culture medium was removed;

[0087] (2) Dilute the EdU solution with complete culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 μmol / L EdU culture medium, add 100 μL of EdU culture medium to each well, and incubate in the incubator for 2 h;

[0088] (3) Discard the culture medium and wash the cells twice with PBS, 5 min each time;

[0089] (4) Add 50 μL of cell fixative to each well, incubate at room temperature for 30 min, and discard the fixative;

[0090] (5) Add 50 μL of 2 mg / mL glycine to each well and incubate on a decolorizing shaker for 5 min;

[0091] (6) Discard glycine and add 100 μL of PBS to wash the cells once;

[0092] (7) Add 100 μL of PBS containing 0.5% TritonX-100 to each well and incubate on a shaker for 10 min;

[0093] (8) Discard the permeabilization agent and wash the cells with PBS for 5 min;

[0094] (9) Add 100 μL of Apollo staining reaction solution to each well and incubate in a shaker at room temperature for 30 min in the dark;

[0095] (10) Discard the staining reaction solution, add 100 μL of 0.5% TritonX-100 in PBS and wash on a shaker three times, 10 min each time;

[0096] (11) Prepare 1× Hoechst33342 reaction solution with deionized water at a ratio of 100:1, add 100 μL of 1× Hoechst33342 reaction solution to each well, and incubate in a light-proof decolorizing shaker at room temperature for 30 min;

[0097] (12) Discard the reaction solution and wash the cells with PBS three times, 5 min each time;

[0098] (13) Imaging analysis was performed using a fluorescence microscope.

[0099] 1.2.5 qRT-PCR detection of gene expression levels

[0100] (1) Primers were designed based on the Gfra1, Ret, Pdx1, C-kit, Sox3, and Rarg gene sequences in GeneBank using the NCBI online tool Pick Primer and synthesized by Xi’an Qingke Jersey Biotechnology Co., Ltd. and Shanghai Shenggong Biotechnology Co., Ltd. The detailed information of the primers is shown in Table 1.

[0101] Table 1 Primer information

[0102]

[0103]

[0104] (2) Total RNA of spermatogonial stem cells cultured at different times was extracted using the Trizol method, the integrity of RNA was detected by electrophoresis, and the concentration of RNA was measured by a nucleic acid analyzer;

[0105] (3) Using a reverse transcription kit One-Step gDNA Removal and cDNASynthesis SuperMix (Quanshijin, AE311-03), to reverse transcribe RNA into cDNA;

[0106] (4) Using β-actin as the internal reference, Top Green qPCR SuperMix (full gold, AQ131-02) kit was used for qPCR amplification. The specific loading volume was: 2×TransStart Top Green qPCRSuperMix 10uL, Nuclease-free Water 8.2μL, PCR upstream primer 0.4μL, PCR downstream primer 0.4μL, template cDNA 1μl, and the total volume was 20μL. Amplification conditions were: 95℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 60℃ annealing for 35s, 72℃ for 15s, 75℃ for 1s, amplification 40 times, 72℃ for 10min;

[0107] (5) Each sample was repeated three times, and the average Ct value of the three samples was calculated. The average Ct value was reset to zero (ΔCt) based on the expression level of the internal reference gene, and the relative expression level of each gene was calculated as 2^-ΔΔCt. The relative expression level is expressed as 2-ΔΔCT.

[0108] 1.3 Results

[0109] 1.3.1 Culture of feeder cells

[0110] (1) Mouse fibroblasts

[0111] Mouse fibroblast cell line was purchased from Beina Biotechnology. MEF cell line was revived by conventional cell culture method. After 48 h of culture, Figure 1 As shown, the cells are in good condition and can be used for subsequent feeder layer cell preparation.

[0112] (2) Tibetan sheep's own supporting cells

[0113] The cells were digested with trypsin and collagenase and purified with Percoll cell separation medium. Cells with 11%-19% and 27%-35% Percoll gradients were cultured and digested with 0.05% trypsin, which is equivalent to a hypotonic solution that can remove the interstitial cells that were not removed in the previous step. After the cells grew for 6 hours, the culture medium was replaced to remove the spermatogenic cells that were not attached to the wall, and testicular supporting cells with good morphological characteristics (fusiform and oval) were obtained. Figure 2 A). Oil Red O and specific GATA4 antibodies were used to identify supporting cells. The results showed that GATA4 protein had a strong fluorescent signal and was distributed in the cytoplasm and nucleus ( Figure 2 B, 2C). The above results show that the isolated primary Tibetan sheep testicular supporting cells have good growth conditions and high purity, and can be used for the subsequent preparation of feeder layers.

[0114] 1.3.2 Isolation, purification and identification of primary spermatogonial stem cells

[0115] Freshly isolated primary testicular cells are transparent and round ( Figure 3 A). The purified spermatogonial stem cells are grape-shaped or mulberry-shaped ( Figure 3 B), which is consistent with the characteristics of spermatogonial stem cells. Further immunofluorescence identification was performed using spermatogonial stem cell marker proteins PLZF and UCLH1. The results showed that both PLZF and UCLH1 proteins had strong fluorescence signals and were distributed in the cytoplasm and nucleus ( Figure 3 C). The above results show that the isolated and purified Tibetan sheep spermatogonial stem cells have good growth conditions and high purity.

[0116] 1.3.3 Culture of spermatogonial stem cells with feeder layer system

[0117] Spermatogonial stem cells were cultured on MEF feeder layer and SCs feeder layer respectively. The results showed that spermatogonial stem cells attached to the two feeder layer cells and formed clones after 7 days of culture. Figure 4 )

[0118] In different feeder layers, the initial growth efficiency of SSCs was similar, but the proliferation efficiency after 14 days was quite different ( Figure 5A), indicating that the proliferation efficiency of SCs as feeder layer is higher than that of MEF. The CCK-8 method was used to determine the effects of different feeder layers on the proliferation of SSCs. The results showed that compared with the MEF feeder layer, the proliferation rate of the SCs feeder layer was higher ( Figure 5 B), indicating that the SCs feeder layer can better promote the proliferation of spermatogonial stem cells, and the proliferation rate at 21 days is more obvious. The EdU labeling method was used to detect the effects of different feeder layers on the proliferation of SSCs. The results showed that compared with the MEF feeder layer, the EdU positive rate of the SCs feeder layer was higher ( Figure 5 C), indicating that the SCs feeder layer can better promote the proliferation of spermatogonial stem cells. The effect of different feeder layers on SSCs proliferation was detected by PLZF protein immunofluorescence. The results showed that compared with the MEF feeder layer, the positive rate of the SCs feeder layer was higher ( Figure 5 D), indicating that the SCs feeder layer can better promote the proliferation of spermatogonial stem cells.

[0119] Furthermore, qRT-PCR experiments were used to detect the effects of different feeder layers on SSCs self-renewal and differentiation marker genes. Among them, Gfra1, Ret and Pdx1 are expressed in undifferentiated spermatogonial stem cells and are generally regarded as spermatogonial stem cell self-renewal marker genes. C-kit, Sox3 and Rarg are related to differentiation and are regarded as differentiation marker genes. The expression of these 6 genes in different feeder layers was detected at different times of culture. The results showed that compared with the MEF feeder layer, the expression levels of Gfra1, Ret and Pdx1 were increased in the supporting cell feeder layer, while the expression levels of C-kit, Sox3 and Rarg were decreased ( Figure 6 ). This indicates that its own SCs as feeder layer can promote the proliferation of spermatogonial stem cells and inhibit their apoptosis.

[0120] 1.3.4 Culture of spermatogonial stem cells without feeder layer system

[0121] Spermatogonial stem cells were cultured in MEMα medium and StemProTM-34SFM medium on laminin-coated culture plates. The results showed that spermatogonial stem cells could proliferate and aggregate in both culture media after 7 days of culture. Figure 7 )

[0122] In different culture media, the initial growth efficiency of SSCs was similar, but the proliferation efficiency after 14 days was quite different. In addition, the clones in StemProTM-34SFM medium were significantly larger and there were fewer cells in the differentiated state ( Figure 8 A). CCK-8 was used to determine the effects of different culture media on SSCs proliferation. The results showed that compared with MEMα culture medium, StemProTM-34SFM culture medium had a higher proliferation rate ( Figure 8B), indicating that StemProTM-34SFM medium can better promote the proliferation of spermatogonial stem cells, and the proliferation rate at 14d and 21d is more obvious. The EdU labeling method was used to detect the effects of different culture media on the proliferation of SSCs. The results showed that compared with MEMα culture medium, the EdU positive rate of StemProTM-34SFM culture medium was higher ( Figure 8 C), indicating that StemProTM-34SFM culture medium can better promote the proliferation of spermatogonial stem cells.

[0123] The effects of different culture media on SSCs proliferation were detected by PLZF protein immunofluorescence. The results showed that compared with MEMα culture medium, the positive rate of spermatogonial stem cells in StemProTM-34SFM culture medium was higher ( Figure 8 D), indicating that StemProTM-34SFM culture medium can better promote the proliferation of spermatogonial stem cells.

[0124] qRT-PCR was used to detect the effects of different culture media on SSCs self-renewal and differentiation marker genes. The results showed that compared with MEMα culture medium, the expression levels of Gfra1, Ret and Pdx1 increased, while the expression levels of C-kit, Sox3 and Rarg decreased in StemProTM-34SFM culture medium ( Fig. 9 ). This indicates that StemProTM-34SFM is more likely to promote the proliferation of spermatogonial stem cells and inhibit their differentiation. StemProTM-34SFM can be used as a basic culture medium for long-term in vitro culture of Tibetan sheep spermatogonial stem cells.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for culturing primary spermatogonial stem cells of Tibetan sheep, characterized in that: The steps include: (1) obtaining testicular tissue of Tibetan sheep lambs, disinfecting and cleaning them, and obtaining tissue blocks; (2) mincing the tissue blocks, digesting, washing, and resuspending to obtain a single cell suspension of the seminiferous tubules; (3) purifying the single cell suspension to obtain Tibetan sheep primary spermatogonial stem cells; (4) culturing the Tibetan sheep primary spermatogonial stem cells without or with a feeder layer; The feeder-free culture is carried out using feeder-free MEMα medium or StemPro TM -34SFM complete medium was cultured in laminin-coated culture vessels; The feeder layer culture is cultured in a mouse fibroblast cell line feeder layer or in a Tibetan sheep primary testicular supporting cell feeder layer, and a feeder layer MEMα culture medium is added.

2. The culture method according to claim 1, characterized in that The preparation method of the primary testicular support cell feeder layer of Tibetan sheep comprises the following steps: collecting the fresh testicular tissue of the Tibetan sheep lamb in step (1), treating it into a sterile tissue block, digesting it with collagenase IV and trypsin in sequence, and after terminating the digestion, sieving the digestion solution step by step to collect the cells; resuspending the cells, separating them with Percoll cell separation solution, collecting the suspended cell solution with 11% to 19% and 27% to 35% Percoll gradients, and purifying them to obtain purified primary testicular support cells of Tibetan sheep; then treating the primary testicular support cells of Tibetan sheep with mitomycin C, and preparing the treated primary testicular support cells of Tibetan sheep into a cell suspension, and culturing the cell suspension into a monolayer of cells in a culture bottle pre-coated with 0.1% gelatin to obtain the primary testicular support cell feeder layer of Tibetan sheep.

3. The culture method according to claim 2, characterized in that The sieves for sieving the digestive fluid step by step are 80-100 meshes, 170-200 meshes and 270-300 meshes respectively; the gradient concentration of the Percoll cell separation fluid is 10-12%, 18-20%, 26-28%, 34-36% and 42-44%.

4. The culture method according to claim 1, characterized in that The feeder-free MEMα culture medium was prepared according to a 50 mL system, and the following components were added: 48-49 mL MEMα culture medium, 0.4-0.6 mL FBS, 0.4-0.6 mL penicillin-streptomycin, 95-105 μL 18-22 ng / mL GDNF, 45-55 μL 9-11 ng / mL bFGF, 45-55 μL 900-1100 U / mL LIF and 45-55 μL 9-11 ng / mL SDF-1.

5. The culture method according to claim 4, characterized in that The StemPro TM -34SFM medium was prepared according to the 50mL system and the following components were added: 44-45mL StemPro TM -34SFM medium, 0.4~0.6mL FBS, 0.4~0.6mL penicillin-streptomycin, 0.4~0.6mL 1% non-essential amino acids, 45~55μL 10μg / mL insulin, 45~55μL 10μg / mL transferrin, 0.4~0.6mL 1% β-mercaptoethanol, 0.4~0.6mL 1% sodium pyruvate, 45~55μL 19~21μg / mL putrescine, 0.4~0.6mL 1% L-glutamine, 0.4~0.6mL 1% vitamins, 0.4~0.6mL 9~11mM Hepes buffer, 0.8~1.2mL 2% StemPro neural supplement, 90~110μL 19~21ng / mL GDNF, 45~55μL 9~11ng / mL bFGF, 45~55μL 1000U / mL LIF and 45~55μL 9~11ng / mL SDF-1.

6. The culture method according to claim 5, characterized in that: The feeder layer MEMα culture medium is prepared according to a 50 mL system, and the following components are added: 48-49 mL MEMα culture medium, 0.4-0.6 mL FBS, 0.4-0.6 mL penicillin-streptomycin, 90-110 μL 19-21 ng / mL GDNF, 48-52 μL 9-11 ng / mL bFGF, 48-52 μL 900-1100 U / mL LIF and 48-52 μL 9-11 ng / mL SDF-1.

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