Tibetan sheep primary spermatogonial stem cell separation, purification and culture method and application thereof

Abstract

The invention relates to the technical field of spermatogonial stem cell separation, in particular to a Tibetan sheep primary spermatogonial stem cell separation, purification and culture method and application thereof. The method comprises the following steps: (1) treating testicular tissues of Tibetan sheep by using mixed enzyme liquid and enzymatic hydrolysate in sequence to obtain single-cell suspension of seminiferous tubules; (2) purifying the single-cell suspension of the seminiferous tubules obtained in the step (1) by using a differential adhesion method to obtain spermatogonial stem cells; and (3) inoculating the spermatogonial stem cells obtained in the step (2) into a feeding layer for culture. The method is simple and easy to implement, the primary spermatogonial stem cells of the Tibetan sheep can be obtained through effective separation and purification, and the purity of the spermatogonial stem cells is high.

Description

Technical Field

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

[0002] Spermatogonia stem cells (SSCs) refer to a type of primitive spermatogonia located on the basement membrane of the seminiferous tubules that can both self-renew and maintain their own proper constant amount and can also differentiate to produce spermatocytes. With the birth of male animals, primordial germ cells transform into gonocytes, migrate to the basement membrane of the seminiferous tubules, and differentiate into spermatogonial stem cells. One spermatogonial stem cell can eventually produce 4096 sperm. In fact, although 75% to 90% of the cells die of apoptosis during differentiation, one spermatogonial stem cell can still produce 400 to 1000 sperm, which shows that spermatogonial stem cells are very efficient in differentiating and producing sperm. From the adolescence of individual animals to old age, spermatogonial stem cells can both self-replicate and enter the spermatogenesis process. After a series of differentiation, they finally produce sperm, and pass the genome to the next generation after combining with the egg. They are regarded as immortal cells because they can both self-replicate in adults and be passed down from generation to generation.

[0003] The isolation, purification and long-term culture of sheep spermatogonial stem cells are important problems that have troubled scholars. Although the long-term culture method of spermatogonial stem cells in mice has been established, due to the existence of racial differences, gene bank differences, and the incompatibility of commercially available antibodies, the long-term culture method of sheep spermatogonial stem cells cannot be copied from the mouse method. There are no successful reports on the isolation, purification, and long-term culture technology operation system of sheep spermatogonial stem cells.

[0004] The current methods for isolating and purifying spermatogonial stem cells mainly include: cryptorchid surgical enrichment, enzymatic hydrolysis, Percoll density gradient centrifugation, flow cytometry sorting, immunomagnetic bead sorting, differential adhesion, etc. Flow cytometry sorting and immunomagnetic bead sorting have good application effects in mouse spermatogonial stem cells, but both methods require antibodies that specifically bind to the surface antigens of spermatogonial stem cells of the species being studied, and antibodies suitable for sheep spermatogonial stem cells are not common. Therefore, flow cytometry sorting and immunomagnetic bead sorting are not suitable for the study of sheep spermatogonial stem cells. Cryptorchid surgical enrichment and Percoll density gradient centrifugation cannot obtain high-purity spermatogonial stem cells, and in practice, it has been found that Percoll density gradient centrifugation has an effect on the vitality of spermatogonial stem cells. So far, there has been no report on the use of these two methods in successful spermatogonial stem cell culture technology.

[0005] At present, some scholars have reported on the successful separation of spermatogonial stem cells using the enzymatic hydrolysis method + differential adhesion method. However, the key to using the enzymatic hydrolysis method + differential adhesion method is that the spermatogonial stem cells contained in the prepared single-cell suspension must be of high purity, large number and good activity. The tissue structure of the testicular spermatogenic epithelial cells of different animals is different. When using the enzymatic hydrolysis method to digest the surrounding tissues and release supporting cells and spermatogonial stem cells, the type and concentration of the digestive enzyme, the action intensity (rotation speed), the digestion time, the buffer composition, etc. are all factors that affect the degree of digestion. If any of the factors is not appropriate, it will affect the digestion of the seminiferous epithelium and the release of spermatogonial stem cells, resulting in a small number of spermatogonial stem cells in the single-cell suspension and poor quality. Finally, purification by the differential adhesion method cannot obtain a sufficient number of spermatogonial stem cells with good activity. Even if a high-quality single-cell suspension is prepared, in the process of purifying spermatogonial stem cells, the specific operating methods such as separation order, separation time, number of washes, and wash intensity are all factors that affect the number and quality of the spermatogonial stem cells collected in the end. If they are not mastered well, spermatogonial stem cells with high activity cannot be obtained. Therefore, although the enzymatic hydrolysis + differential adhesion method is relatively simple in principle, and the instruments and reagents used are also relatively common, the different species and different requirements of each operation link will directly affect the separation and purification effect of spermatogonial stem cells. As a result, there has been no successful report on the method of successfully separating and purifying sheep spermatogonial stem cells using the enzymatic hydrolysis + differential adhesion method.

[0006] Therefore, how to provide a method for separating, purifying and culturing Tibetan sheep primary spermatogonial stem cells to solve the technical problems in the prior art that primary spermatogonial stem cells in Mianyang are difficult to separate, the purification and culture methods are complicated, and the effect is poor is an issue that technical personnel in this field urgently need to solve. Summary of the invention

[0007] The purpose of the present invention is to provide a method for separating, purifying and culturing primary spermatogonial stem cells of Tibetan sheep and its application. The method of the present invention is simple and easy to implement, and can effectively separate and purify primary spermatogonial stem cells of Tibetan sheep, and the spermatogonial stem cells have high purity.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for separating, purifying and culturing primary spermatogonial stem cells of Tibetan sheep, comprising the following steps:

[0010] (1) sequentially treating Tibetan sheep testicular tissue with a mixed enzyme solution and an enzymatic hydrolysate to obtain a single cell suspension of seminiferous tubules; the mixed enzyme solution is one, two or more of collagenase, DNase I enzyme, hyaluronidase and Dispase degrading enzyme; the enzymatic hydrolysate is pancreatic enzyme and / or DNase I enzyme;

[0011] (2) purifying the single cell suspension of the seminiferous tubules obtained in step (1) using a differential adhesion method to obtain spermatogonial stem cells;

[0012] (3) The spermatogonial stem cells obtained in step (2) are inoculated into a feeder layer for culture.

[0013] Preferably, the medium used in the differential adhesion method in step (2) is gelatin with a concentration of 0.08-0.12%, gelatin with a concentration of 0.18-0.22%, or poly-lysine.

[0014] Preferably, in step (1), when the mixed enzyme solution is collagenase and DNaseⅠ, the enzymatic solution is pancreatic enzyme and DNaseⅠ enzyme;

[0015] When the mixed enzyme solution contains collagenase, hyaluronidase, and DNaseⅠ, the enzymatic solution is pancreatic enzyme;

[0016] When the mixed enzyme solution includes collagenase, Dispase degrading enzyme, and DNaseⅠ enzyme, the enzymatic solution is selected as pancreatic enzyme.

[0017] Preferably, in the mixed enzyme solution of step (1), the concentration of collagenase is 0.25-2.00 mg / mL; the concentration of DNaseⅠ is 20 μg / mL-7 mg / mL; the concentration of hyaluronidase is 1-3 mg / mL; and the concentration of Dispase is 1-3 mg / mL.

[0018] Preferably, in the enzymatic hydrolysis solution of step (1), the concentration of pancreatic enzyme is 0.15-0.35% (V / V); the concentration of DNase I enzyme is 5-9 mg / mL.

[0019] Preferably, the concentration of poly-lysine used in step (2) is 0.5-1.5 mg / mL; and the number of differential attachments in the differential attachment method is 1-3 times.

[0020] Preferably, the differential adhesion method in step (2) is: using poly-lysine at a concentration of 0.5 to 1.5 mg / mL as a medium, wrapping the bottom of the culture bottle, discarding the liquid after cultivation, rinsing, then adding culture medium and inoculating a single cell suspension of the seminiferous tubules, collecting the cell suspension after cultivation, and centrifuging.

[0021] Preferably, the differential adhesion method in step (2) is as follows: 1) using gelatin with a concentration of 0.08-0.12%, gelatin with a concentration of 0.18-0.22% or poly-lysine as a medium, wrapping the bottom of the culture bottle, discarding the liquid after cultivation, rinsing, then adding culture medium and inoculating a single cell suspension of seminiferous tubules, collecting the cell suspension after cultivation, centrifuging, and obtaining a purified single cell suspension of seminiferous tubules; 2) taking the purified single cell suspension of seminiferous tubules and repeating the method of step 1) twice.

[0022] Preferably, the differential adhesion method in step (2) is as follows: Percoll working solution and the single cell suspension of the seminiferous tubules are mixed, centrifuged and the supernatant is discarded, after washing, centrifuged and the supernatant is discarded to obtain a cell suspension; the bottom of the culture bottle is wrapped with gelatin with a concentration of 0.08-0.12% as a medium, the liquid is discarded after cultivation, and then rinsed, and then the culture medium is added and the single cell suspension of the seminiferous tubules is inoculated, and the cell suspension is collected after cultivation and centrifuged.

[0023] The present invention also provides the use of the Tibetan sheep primary spermatogonial stem cells prepared by the method in Tibetan sheep assisted breeding.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention adopts a two-step enzyme combined with DnaseⅠ enzyme digestion method in the separation process of spermatogonial stem cells, which is simple and convenient to operate and has a higher proportion of spermatogonial stem cells in the obtained single cell suspension; the single cell suspension obtained by separation is further subjected to 30% percoll centrifugation and 0.1% gelatin differential adhesion for 24 hours to obtain spermatogonial stem cells with a purity of more than 80%. Compared with other methods, this method is simple to operate, has low required cost, and the purity of purified spermatogonial stem cells is higher, which meets the requirements of cell operations such as cell transfection and genetic modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0027] Figure 1 This is the morphological characteristics of primary testicular cells in Experimental Example 1.

[0028] Figure 2 The cell positive rate of spermatogonial stem cells isolated by different methods in Experimental Example 2; A: (a) 0.25mg / mL collagenase IV + 7mg / mL DnaseⅠ enzyme; (b) 0.25% pancreatic enzyme + 7mg / mL DnaseⅠ enzyme; B: (a) 2mg / mL collagenase IV + 2mg / mL hyaluronidase + 80ug / mL DnaseⅠ enzyme; (b) 0.25% pancreatic enzyme; C: (a) 2mg / mL collagenase IV + 2mg / mL Dispase decomposition enzyme + 20ug / mL DnaseⅠ enzyme; (b) 0.25% pancreatic enzyme.

[0029] Figure 3This is the purification of spermatogonial stem cells in Experimental Example 3; (A) After the cell suspension was centrifuged in 30% percoll solution, it was inoculated on a culture plate incubated with 0.1% gelatin; (B) The cell suspension was inoculated on a culture plate incubated with 0.1% gelatin.

[0030] Figure 4 This is the cell positive rate of purifying spermatogonial stem cells using the differential adhesion method using different media in Experimental Example 4; A: 0.1% gelatin differential adhesion for 24 hours; B: 0.2% gelatin differential adhesion for 24 hours; C: poly-lysine differential adhesion for 24 hours.

[0031] Figure 5 For Experimental Example 5, 0.1% gelatin was used to purify testicular primary cells multiple times; A: (a) 24 h of patching; B: (a) 6 h of patching; (b) 24 h of patching; C: (a) 2 h of patching; (b) 4 h of patching; (c) 24 h of patching.

[0032] Figure 6 This is the cell positive rate of purifying spermatogonial stem cells using the differential adhesion method and the percoll separation method in Experimental Example 6; A: 0.1% gelatin differential adhesion for 24 hours; B: 0.1% gelatin differential adhesion for 24 hours combined with 30% percoll centrifugation.

[0033] Figure 7 For Experimental Example 7, alkaline phosphatase staining was used to identify spermatogonial stem cells.

[0034] Figure 8 Experimental Example 7: Immunofluorescence staining to identify spermatogonial stem cells: scale bar 50 μm.

[0035] Fig. 9 This is Experimental Example 8: Spermatogonial stem cell proliferation culture. DETAILED DESCRIPTION

[0036] The present invention provides a method for separating, purifying and culturing primary spermatogonial stem cells of Tibetan sheep, comprising the following steps:

[0037] (1) sequentially treating Tibetan sheep testicular tissue with a mixed enzyme solution and an enzymatic hydrolysate to obtain a single cell suspension of seminiferous tubules; the mixed enzyme solution is one, two or more of collagenase, DNase I enzyme, hyaluronidase and Dispase degrading enzyme; the enzymatic hydrolysate is pancreatic enzyme and / or DNase I enzyme;

[0038] (2) purifying the single cell suspension of the seminiferous tubules obtained in step (1) using a differential adhesion method to obtain spermatogonial stem cells;

[0039] (3) The spermatogonial stem cells obtained in step (2) are inoculated into a feeder layer for culture.

[0040] In the present invention, the medium used in the differential adhesion method in step (2) is gelatin with a concentration of 0.08-0.12%, gelatin with a concentration of 0.18-0.22%, or poly-lysine.

[0041] In the present invention, in step (1), when the mixed enzyme solution is collagenase and DNaseⅠ, the enzymatic solution is selected from pancreatic enzyme and DNaseⅠ enzyme;

[0042] When the mixed enzyme solution contains collagenase, hyaluronidase, and DNaseⅠ, the enzymatic solution is pancreatic enzyme;

[0043] When the mixed enzyme solution includes collagenase, Dispase degrading enzyme, and DNaseⅠ enzyme, the enzymatic solution is selected as pancreatic enzyme.

[0044] In the present invention, the concentration of collagenase in the mixed enzyme solution of step (1) is 0.25-2.00 mg / mL; preferably 1-1.4 mg / mL; more preferably 1.1-1.3 mg / mL; and more preferably 1.2 mg / mL.

[0045] In the present invention, in the mixed enzyme solution of step (1), the concentration of DNaseⅠ enzyme used is 20 μg / mL to 7 mg / mL; preferably 1 mg / mL to 5 mg / mL; more preferably 2 mg / mL to 4 mg / mL; and more preferably 3 mg / mL.

[0046] In the present invention, the concentration of hyaluronidase in the mixed enzyme solution of step (1) is 1 to 3 mg / mL, preferably 2 mg / mL.

[0047] In the present invention, in the mixed enzyme solution of step (1), the concentration of Dispase is 1-3 mg / mL, preferably 2 mg / mL.

[0048] In the present invention, in the enzymatic hydrolysis solution of step (1), the concentration of pancreatic enzyme used is 0.15-0.35% (V / V); preferably, more preferably, more preferably.

[0049] In the present invention, in the enzymatic hydrolysis solution of step (1), the concentration of DNase I enzyme is 5 to 9 mg / mL; preferably 6 to 8 mg / mL; more preferably 7 mg / mL.

[0050] In the present invention, the concentration of poly-lysine used in step (2) is 0.5-1.5 mg / mL; preferably 0.7-1.3 mg / mL; more preferably 0.9-1.1 mg / mL; and more preferably 1 mg / mL.

[0051] In the present invention, the number of times of differential adhesion in step (2) is 1 to 3 times, preferably 2 times.

[0052] In the present invention, the differential adhesion method in step (2) is as follows: using 0.08-0.12% gelatin, 0.18-0.22% gelatin or 0.5-1.5 mg / mL poly-lysine as a medium, wrapping the bottom of the culture bottle, discarding the liquid after cultivation, rinsing, then adding culture medium and inoculating a single cell suspension of the seminiferous tubules, collecting the cell suspension after cultivation, and centrifuging.

[0053] In the present invention, the medium preferably has a concentration of 0.09-0.11% gelatin, 0.19-0.21% gelatin and 0.7-1.3 mg / mL polylysine; further preferably has a concentration of 0.1% gelatin, 0.2% gelatin and 0.9-1.1 mg / mL polylysine; more preferably has a concentration of 0.1% gelatin, 0.2% gelatin and 1 mg / mL polylysine.

[0054] In the present invention, the differential adhesion method in step (2) is as follows: 1) using gelatin with a concentration of 0.08-0.12% as a medium, wrapping the bottom of the culture bottle, discarding the liquid after cultivation, rinsing, then adding culture medium and inoculating a single cell suspension of seminiferous tubules, collecting the cell suspension after cultivation, centrifuging, and obtaining a purified single cell suspension of seminiferous tubules; 2) taking the purified single cell suspension of seminiferous tubules and repeating the method of step 1) twice.

[0055] In the present invention, the medium is preferably gelatin with a concentration of 0.09-0.11%, and more preferably 0.1% gelatin.

[0056] In the present invention, the differential adhesion method in step (2) is as follows: Percoll working solution and single cell suspension of seminiferous tubules are mixed, centrifuged and supernatant is discarded, washed and centrifuged and supernatant is discarded to obtain a cell suspension; polylysine with a concentration of 0.5 to 1.5 mg / mL is used as a medium to wrap the bottom of the culture bottle, discard the liquid after cultivation, rinse, then add culture medium and inoculate the single cell suspension of seminiferous tubules, collect the cell suspension after cultivation, and centrifuge.

[0057] The present invention also provides the use of the Tibetan sheep primary spermatogonial stem cells prepared by the method in Tibetan sheep assisted breeding.

[0058] 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.

[0059] Materials used in the experiment:

[0060] 1. Test Materials

[0061] One month old Tibetan sheep lamb

[0062] 2. Test Preparation

[0063] Sterile scissors, forceps, clean bench, 75% ethanol, penicillin-streptomycin (gibco, 15140163), PBS buffer (Solerabol, P1020), HBSS buffer (gibco, 14025092), T25 culture flask (Corning, CLS430639), 6-well plate (costar, 3516), 0.25% trypsin (gibco, 15050065), collagenase type IV (sigma, C5138), hyaluronidase (Sigma, H3884), DNaseⅠ enzyme (Sigma, 10104159001) , Dispase (Yeasen, 40104ES60), 1% gelatin (Solebol, G0040), 1 mg / mL poly-lysine (Harling Biotechnology, HLH0095), percoll cell separation solution (Sigma, P1644), red blood cell lysis solution (Solebol, R1010), MEMα culture medium (gibco, 41061029), fetal bovine serum (gibco, 10099-141C), alkaline phosphatase staining kit (Solebol, G1480), mitomycin C (Aladdin, D419552), Rabbit Anti-Plzf antibody (abcam, ab-104854), Rabbit Anti-UCHL1+3antibody (Boaosen, bs-11677R), Rabbit Anti-CD90 / Thy-1antibody (Boaosen, bs-0778R), GoatAnti-Rabbit IgG H&L antibody (Boaosen, bs-0295G-Cy3).

[0064] 3. Reagent Preparation

[0065] ① Type IV collagenase (sigma, C5138)

[0066] a. Weigh 20 mg of type IV collagenase and dissolve it in 10 mL of PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare a 2 mg / mL cell digestion solution;

[0067] b. 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;

[0068] ②DNaseⅠ enzyme (Sigma, 10104159001)

[0069] a. Weigh 175 mg of DNaseⅠ enzyme and dissolve it in 25 mL of PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare a 7 mg / mL cell digestion solution;

[0070] b. Weigh 0.8 mg of DNaseⅠ enzyme and dissolve it in 10 mL of PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare 80 μg / mL cell digestion solution;

[0071] c. Weigh 0.2 mg of DNaseⅠ enzyme and dissolve it in 10 mL of PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare a 20 μg / mL cell digestion solution;

[0072] ③ Hyaluronidase (Sigma, H3884): Weigh 20 mg of hyaluronidase and dissolve it in 10 mL of PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare a 2 mg / mL cell digestion solution;

[0073] ④ Dispase (Yeasen, 40104ES60): Weigh 20 mg of Dispase and dissolve it in 10 mL of PBS without calcium and magnesium. After fully dissolved, filter it with a 0.22 μm filter to prepare a 2 mg / mL cell digestion solution;

[0074] ⑤ Gelatin working solution

[0075] a. Take 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;

[0076] b. Pipette 2 mL of 1% gelatin solution and add it to 8 mL of PBS without calcium and magnesium to dilute it into 0.2% gelatin solution;

[0077] ⑥ Preparation of complete culture medium: MEMα culture medium (gibco, 41061029), 1% FBS (gibco, 10099-141C), 1% penicillin-streptomycin (gibco, 15140163), 20 ng / mL GDNF, 10 ng / mL bFGF, 1000 U / mL LIF, 10 ng / mL SDF-1.

[0078] Example 1

[0079] A method for separating testicular tissue, comprising the following steps:

[0080] 1. Take lamb testicular tissue and place it in 75% ethanol for rapid disinfection for 1 minute;

[0081] 2. Wash three times in PBS containing 1% PS in a clean bench;

[0082] 3. Use sterile scissors to cut off the external white membrane and epididymis of the testis, then cut it in the middle avoiding the external blood vessels of the testis, take about 200 mg of tissue blocks from the middle of each testis, transfer the tissue blocks into a culture dish containing HBSS, and cut them into small pieces with scissors to obtain testicular tissue.

[0083] Example 2

[0084] A method for preparing a single cell suspension of seminiferous tubules, comprising the following steps:

[0085] Add 20mL 0.25mg / mL type IV collagenase and 1mL 7mg / mL DNaseⅠ to a 50mL centrifuge tube, transfer 400mg of the shredded tissue obtained in Example 1 into the tube and digest for 7min. Transfer the digestion solution to a 15mL centrifuge tube, and place the tube in an ice box for 2min and discard the supernatant. Add 3mL HBSS buffer and wash 5 times. For the last wash, centrifuge at 300g for 1min at 4℃ and discard the supernatant. Add 8mL 0.25% trypsin and 2mL DNaseⅠ, mix well and place in a 37℃ water bath for 25min, shaking every 5min. Add 1.5mL DMEM / F-12 culture medium and 0.5mL DNaseⅠ to the above tube, centrifuge at 600g for 7min at 4℃ and discard the supernatant. Add 5 mL DNaseⅠ and 2 mL HBSS, mix well with a pipette tip, and then pour it into a 100 μm cell sieve for filtration. Centrifuge the cell suspension at 600 g at 4°C for 7 min and discard the supernatant. Wash the precipitate twice with 5 mL HBSS to obtain a single-cell suspension of seminiferous tubules with a concentration of 29.2±0.9%.

[0086] Example 3

[0087] A method for preparing a single cell suspension of seminiferous tubules, comprising the following steps:

[0088] Add HBSS to a 15mL centrifuge tube, mix 400mg of the tissue block obtained in Example 1, centrifuge at 1800rpm 4℃ for 5min, and remove the supernatant. Repeat this step 3 times. Add 3mL 2mg / mL type IV collagenase, 3mL 2mg / mL hyaluronidase and 3mL 80μg / mL DNaseⅠ enzyme, digest in a 37℃ water bath for 25min, and shake once every 5min. Centrifuge the digestion solution at 1800rpm 4℃ for 5min and discard the supernatant to obtain testicular tissue fragments. Add HBSS to the centrifuge tube with testicular tissue fragments, shake the centrifuge tube to mix it evenly, let the centrifuge tube stand, let it settle naturally for 2min, take the supernatant, and repeat 3 times. Centrifuge the supernatant at 1800rpm 4℃ for 5min and discard the supernatant to obtain seminiferous tubule fragments. Add 2mL of red blood cell lysis buffer to the seminiferous tubule fragments and resuspend them with a pipette, centrifuge the cell suspension at 1500rpm 4℃ for 5min and discard the supernatant. Add 2 mL of 0.25% trypsin and mix thoroughly. Digest in a 37°C metal bath for 5 minutes and then examine under a microscope (no obvious tubular appearance is observed in the field of view, and the cells are evenly dispersed). Add DMEM / F-12 medium to terminate digestion, centrifuge at 1500 rpm and 4°C for 5 minutes, and discard the supernatant. Add DMEM / F-12 medium containing 5% FBS to resuspend, filter through a 40 μm cell sieve, and obtain a single cell suspension of seminiferous tubules with a concentration of 19.1±2.0%.

[0089] Example 4

[0090] A method for preparing a single cell suspension of seminiferous tubules, comprising the following steps:

[0091] ③ Add HBSS to a 15mL centrifuge tube, mix 400mg of the tissue block obtained in Example 1, centrifuge at 1800rpm at 4℃ for 5min, and remove the supernatant. Repeat this step 3 times. Add 2mg / mL IV collagenase, 2mg / mL Dispase decomposition enzyme, 20μg / mL DNaseⅠ enzyme (volume of testicular tissue: volume of IV collagenase: volume of hyaluronidase: volume of DNaseⅠ enzyme = 1:3:3:3), digest in a 37℃ water bath for 25min, and shake once for 5min. Centrifuge the digestion solution at 1200rpm at 4℃ for 5min, and discard the supernatant to obtain testicular tissue fragments. Add HBSS to the centrifuge tube, shake the centrifuge tube to mix it evenly, let the centrifuge tube stand, let it settle naturally for 2min, take the supernatant, and repeat 3 times. Centrifuge the supernatant at 1200rpm at 4℃ for 5min, and discard the supernatant to obtain fragments of seminiferous tubules. Add red blood cell lysis buffer to the seminiferous tubule fragments at a volume ratio of 1:3, resuspend with a pipette and act for 2 minutes, add an equal volume of HBSS to stop, centrifuge at 1200rpm for 5 minutes at 4°C, and discard the supernatant. Wash again with HBSS until there are no red blood cells. Add 0.25% trypsin according to a volume ratio of 1:5, resuspend to mix thoroughly, and digest at 37°C for 5 minutes. Add DMEM / F-12 medium containing 10% FBS to stop digestion, sieve through a 40μm cell sieve, and obtain a single cell suspension of seminiferous tubules with a concentration of 18.5±1.8%.

[0092] Example 5

[0093] A method for purifying a single cell suspension of seminiferous tubules, comprising the following steps:

[0094] 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. Before the culture flask is dried, take 5×10 cells obtained in Example 2 5 The cells were inoculated into a culture flask, and DMEM / F-12 medium containing 5% FBS was added. The cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. The cell suspension was collected and centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded to obtain SSCs with a purity of 61.9±2.8%.

[0095] Example 6

[0096] A method for purifying a single cell suspension of seminiferous tubules, comprising the following steps:

[0097] 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. Before the culture flask is dried, take 5×10 cells obtained in Example 2 5Inoculate cells into a culture flask, add DMEM / F-12 medium containing 5% FBS, and culture in a 37℃5% CO2 incubator. After 2 hours of culture, perform the first differential adhesion. Centrifuge the cell suspension, resuspend it with DMEM / F-12 medium containing 5% FBS, transfer it to a new culture flask coated with 0.1% gelatin in advance and continue to culture. After 4 hours of culture, perform the second differential adhesion. After another 24 hours of culture, perform the third differential adhesion. Collect the cell suspension, centrifuge it at 1200rpm for 5 minutes, and discard the supernatant to obtain SSCs with a purity of 67.4±2.3%.

[0098] Example 7

[0099] A method for purifying a single cell suspension of seminiferous tubules, comprising the following steps:

[0100] Add 2 mL of 30% percoll working solution to a 15 mL centrifuge tube, then add 5 mL of the cell suspension obtained in Example 2, mix well, centrifuge at 600 g for 8 min at 4°C, and discard the supernatant. Add 3 mL of HBSS to the tube for washing, centrifuge at 600 g for 7 min at 4°C, and discard the supernatant. 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. Take 5×10 cells obtained in Example 2 before the culture flask is dried. 5 The cells were inoculated into a culture flask, and DMEM / F-12 medium containing 5% FBS was added. The cells were cultured in a 37°C 5% CO2 incubator for 24 hours, and then the cell suspension was centrifuged and resuspended with DMEM / F-12 medium containing 5% FBS. After further culture for 24 hours, the cell suspension was collected and centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded to obtain SSCs with a purity of 80.5±7.4%.

[0101] Example 8

[0102] A method of culturing spermatogonial stem cells comprises the following steps:

[0103] 5. Prepare feeder layer cells: Prepare mouse embryonic fibroblasts used as feeder layer in advance. Add 0.5mg / mL 100μl mitomycin C to treat cells in T25 culture flasks and incubate in a 37°C incubator for 3h. After incubation, aspirate the culture medium and rinse the wells three times with HBSS. Add 0.25% trypsin to digest the cells and incubate at 37°C for 3min. Add 2mL of culture medium to each flask to terminate digestion. Aspirate the cell suspension into a centrifuge tube and centrifuge at 800rpm for 5min. Discard the supernatant and resuspend the cells in the culture medium. Count the cells and take 2×10 5The cells were cultured in a 0.1% gelatin pre-coated culture flask. Within 24-48 hours, the cells formed a monolayer at the bottom of the culture flask, which was used as a feeder layer for spermatogonial stem cells.

[0104] 6. Remove the culture medium in the feeder layer and wash with HBSS 3 to 4 times. Repeatedly blow and collect the purified spermatogonial stem cells by centrifugation. After resuspending in the culture medium, count the cells and take 3×10 5 Inoculate 100 cells on the feeder layer, add 5 mL of complete medium, and place the cells in an incubator at 37°C and 5% CO2 for culture.

[0105] Experimental Example 1

[0106] Primary spermatogonial stem cells were isolated according to the method of Example 2. Figure 1 As shown. The newly isolated primary testicular cells are transparent and round ( Figure 1 A). After 2 days of culture, some cells adhered to the wall, and the suspended cells aggregated ( Figure 1 B) After 4 days of culture, most of the adherent cells are spindle-shaped supporting cells ( Figure 1 C,D).

[0107] Experimental Example 2

[0108] Primary spermatogonial stem cells were isolated according to the methods of Example 2, Example 3, and Example 4, respectively, and immunofluorescence staining was performed using spermatogonial stem cell-specific marker antibodies ( Figure 2 ). The results showed that method A (Example 2) was more effective in isolating spermatogonial stem cells.

[0109] The specific method of immunofluorescence staining is as follows:

[0110] (1) Use a PAP PEN 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;

[0111] (2) Add 4% paraformaldehyde for 15 min;

[0112] (3) Wash with PBS three times, 5 min each time;

[0113] (4) Add 0.3% TritonX-100 for permeabilization for 10 min;

[0114] (5) Wash with PBS three times, 5 min each time;

[0115] (6) Add blocking solution and block at room temperature for 60 min;

[0116] (7) Add primary antibodies CD90 (1:200), UCHL1 (1:200), and PLZF (1:200) and incubate at 4°C overnight;

[0117] (8) Wash with PBST three times, 5 min each time;

[0118] (9) Add goat anti-rabbit secondary antibody (1:200) and incubate at room temperature in the dark for 1 h;

[0119] (10) Wash with PBST three times, 5 min each time;

[0120] (11) DAPI solution was added to stain the cell nuclei and incubated at room temperature in the dark for 10 min;

[0121] (12) Wash with PBST three times, 5 min each time;

[0122] (13) Observe the cells and take photos under a confocal microscope.

[0123] Experimental Example 3

[0124] According to the separation method of Example 2, the cells were purified using the purification methods of Example 7 and Example 6, respectively. The results are as follows: Figure 3 As shown: It was found that the morphology of these purified cells was similar to that of spermatogonial stem cells.

[0125] Experimental Example 4

[0126] Investigate the effects of different media on purification effects;

[0127] The methods of Example 2 and Example 5 were used as Experimental Group 1;

[0128] Control group 1: other methods were the same as those in Example 5, except that the medium was replaced with an equal amount of 0.2% gelatin solution;

[0129] Control group 2: other methods were the same as those in Example 5, except that the medium was replaced with an equal amount of 1 mg / mL poly-lysine solution;

[0130] The results showed that the purity of spermatogonial stem cells purified by 0.1% gelatin differential adhesion for 24 hours was above 61%, the purity of spermatogonial stem cells purified by differential adhesion for 24 hours was above 55%, and the purity of spermatogonial stem cells purified by poly-lysine differential adhesion for 24 hours was above 52% ( Figure 4 ). Therefore, the results of immunofluorescence staining showed that the spermatogonial stem cells purified using 0.1% gelatin had a higher purity.

[0131] Experimental Example 5

[0132] To investigate the effect of the number of differential adhesion on the purification effect;

[0133] The methods of Example 2 and Example 6 were used as Experimental Group 2;

[0134] Control group 3: other methods were the same as those in Example 6, except that the culture time was 6 h and differential attachment was performed only once.

[0135] Control group 4, other methods are the same as Example 6, the only difference is that the differential attachment is performed twice, the first differential attachment is performed after 6 hours of culture, and the second differential attachment is performed after 24 hours of culture.

[0136] The results showed that the purity of purified spermatogonial stem cells was above 61% when 0.1% gelatin was used for differential attachment once for 24 hours; the purity of purified spermatogonial stem cells was above 63% when 0.1% gelatin was used for differential attachment twice for 6 hours and 24 hours respectively; the purity of purified spermatogonial stem cells was above 67% when 0.1% gelatin was used for differential attachment three times for 2 hours, 4 hours and 24 hours respectively. Figure 5 ). The results of immunofluorescence staining showed that spermatogonial stem cells that were differentially adhered three times had higher purity.

[0137] Experimental Example 6

[0138] The purity of the SSCs obtained by the methods of Example 6 and Example 7 was investigated. The results showed that the purity of the cells obtained by the method of Example 6 was above 61%, and the purity of the spermatogonial stem cells purified by the method of Example 7 was above 80% ( Figure 6 ). Therefore, the immunofluorescence staining results show that the spermatogonial stem cells purified by the method of Example 7 have a higher purity.

[0139] Experimental Example 7

[0140] Spermatogonial stem cells were separated and purified according to the methods of Example 2 and Example 5, respectively, and the purified spermatogonial stem cells were stained with alkaline phosphatase. The results showed that the purified spermatogonial stem cells were stained purple or blue-purple. Alkaline phosphatase staining showed that the purified cells were spermatogonial stem cells ( Figure 7 ).

[0141] Specific staining method: Stain according to the instructions of the alkaline phosphatase staining kit. Centrifuge the cell suspension at 1200rpm for 5min, resuspend with 1mL PBS and inoculate in a six-well plate, and bake in a 60℃ oven for 30min. Add ALP fixative to fix for 3min, and wash with distilled water. Add ALP incubation solution, place in a wet box, incubate in the dark for 20min, and wash with distilled water. Add nuclear fast red staining solution or methyl green staining solution to counterstain for 5min. Wash with PBS and examine under a microscope.

[0142] Immunofluorescence staining was performed using spermatogonial stem cell-specific marker antibodies PLZF, CD90, and UCHL1. The results showed that spermatogonial stem cells showed red fluorescence. Therefore, the immunofluorescence staining results further showed that the purified cells were spermatogonial stem cells ( Figure 8 ).

[0143] Experimental Example 8

[0144] Spermatogonial stem cells were isolated, purified and cultured according to the methods of Example 2, Example 5 and Example 8, and the spermatogonial stem cells were placed in the MEF feeder layer for culture. The results showed that spermatogonial stem cells can proliferate and produce classic grape-like clones ( Fig. 9 ).

[0145] 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 separating, purifying and culturing primary spermatogonial stem cells of Tibetan sheep, characterized in that: The steps include: (1) sequentially treating Tibetan sheep testicular tissue with a mixed enzyme solution and an enzymatic hydrolysate to obtain a single cell suspension of seminiferous tubules; the mixed enzyme solution is one, two or more of collagenase, DNase I enzyme, hyaluronidase and Dispase degrading enzyme; the enzymatic hydrolysate is pancreatic enzyme and / or DNase I enzyme; (2) purifying the single cell suspension of the seminiferous tubules obtained in step (1) using a differential adhesion method to obtain spermatogonial stem cells; (3) The spermatogonial stem cells obtained in step (2) are inoculated into a feeder layer for culture.

2. The method according to claim 1, characterized in that The medium used in the differential adhesion method in step (2) is gelatin with a concentration of 0.08-0.12%, gelatin with a concentration of 0.18-0.22%, or poly-lysine.

3. The method according to claim 1, characterized in that Step (1) when the mixed enzyme solution is collagenase and DNaseⅠ, the enzymatic solution is selected from pancreatic enzyme and DNaseⅠ enzyme; When the mixed enzyme solution contains collagenase, hyaluronidase, and DNaseⅠ, the enzymatic solution is pancreatic enzyme; When the mixed enzyme solution includes collagenase, Dispase and DNaseⅠ, the enzymatic solution is pancreatic enzyme.

4. The method according to claim 1, characterized in that: In the mixed enzyme solution of step (1), the concentration of collagenase is 0.25-2.00 mg / mL; the concentration of DnaseⅠ is 20 μg / mL-7 mg / mL; the concentration of hyaluronidase is 1-3 mg / mL; and the concentration of Dispase is 1-3 mg / mL.

5. The method according to claim 1 or 3, characterized in that: In the enzymatic hydrolysis solution of step (1), the concentration of pancreatic enzyme is 0.15-0.35% (V / V); the concentration of DNaseⅠ enzyme is 5-9 mg / mL.

6. The method according to claim 2, characterized in that The concentration of poly-lysine used in step (2) is 0.5-1.5 mg / mL; the number of differential attachments in the differential attachment method is 1-3 times.

7. The method according to claim 2, characterized in that The differential adhesion method in step (2) is as follows: using gelatin with a concentration of 0.08-0.12%, gelatin with a concentration of 0.18-0.22% or poly-lysine as a medium, wrapping the bottom of the culture bottle, discarding the liquid after cultivation, rinsing, then adding culture medium and inoculating a single cell suspension of the seminiferous tubules, collecting the cell suspension after cultivation, and centrifuging.

8. The method according to claim 2, characterized in that: The differential adhesion method in step (2) is as follows: 1) using gelatin with a concentration of 0.08-0.12% as a medium to wrap the bottom of the culture bottle, discarding the liquid after cultivation, rinsing, then adding culture medium and inoculating a single cell suspension of seminiferous tubules, collecting the cell suspension after cultivation, centrifuging, and obtaining a purified single cell suspension of seminiferous tubules; 2) taking the purified single cell suspension of seminiferous tubules and repeating the method of step 1) twice.

9. The method according to claim 2, characterized in that: The differential adhesion method in step (2) is as follows: Percoll working solution and single cell suspension of seminiferous tubules are mixed, centrifuged and supernatant discarded, washed and centrifuged, and supernatant discarded to obtain cell suspension; gelatin with a concentration of 0.08-0.12% is used as a medium to wrap the bottom of the culture bottle, the liquid is discarded after cultivation, and then rinsed, and then culture medium is added and single cell suspension of seminiferous tubules is inoculated, and the cell suspension is collected after cultivation and centrifuged.

10. Use of the Tibetan sheep primary spermatogonial stem cells obtained by the method according to any one of claims 1 to 9 in Tibetan sheep assisted breeding.

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