Tibetan sheep primary interstitial cell separation and purification method

Abstract

The invention relates to the technical field of interstitial cell separation and purification, in particular to a Tibetan sheep primary interstitial cell separation and purification method. The invention provides a Tibetan sheep primary interstitial cell separation and purification method which comprises the following steps: (1) mixing and digesting lamb testicular tissues, IV type collagenase and bovine serum albumin, centrifuging and discarding supernatant to obtain zymolyte; (2) carrying out mixed digestion on the zymolyte and trypsin, filtering, centrifuging, discarding the supernatant, and washing to obtain a testis single-cell suspension; and (3) primarily culturing the testis single-cell suspension, centrifuging, discarding the supernatant, and precipitating and separating the obtained cells to obtain the testis primary interstitial cells. According to the method, a large number of high-purity testis primary interstitial cells can be rapidly obtained, and the method is simple, convenient and easy to implement, good in repeatability, low in cost and suitable for large-scale application and popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of interstitial cell isolation and purification, and particularly to a method for isolating and purifying primary interstitial cells of Tibetan sheep. Background Art

[0002] As an important livestock resource in the Qinghai-Tibet Plateau region, Tibetan sheep (Ovis aries) have unique physiological characteristics such as cold tolerance, hypoxia tolerance, and adaptation to high-altitude environments. Its mesenchymal stem cells (MSCs), as a type of pluripotent stem cells, have broad application prospects in the fields of tissue engineering, regenerative medicine, and disease model construction. Mesenchymal stem cells have the abilities of self-renewal, multi-directional differentiation potential (such as differentiating into osteoblasts, adipocytes, chondrocytes, etc.), and immunomodulatory functions, thus becoming an important tool for cell therapy and basic research.

[0003] However, the isolation and purification of Tibetan sheep interstitial cells face many challenges. First, it is relatively difficult to obtain tissue samples of Tibetan sheep, especially in plateau areas where experimental conditions are limited. Second, the content of interstitial cells in tissues is low, and they are easily mixed with other cells (such as fibroblasts, endothelial cells, etc.), resulting in insufficient isolation purity. In addition, traditional isolation methods (such as enzyme digestion method, adherent screening method, etc.) may have problems such as cell activity damage, low efficiency, or low purity.

[0004] Currently, the commonly used methods for isolating interstitial cells mainly include:

[0005] Enzyme digestion method: Using collagenase, trypsin, etc. to digest tissues and release cells, but it may cause damage to cell membranes.

[0006] Adherent screening method: Using the adherent characteristics of interstitial cells for screening, but it takes a long time and the purity is limited.

[0007] Density gradient centrifugation method: Separating cells with different densities by centrifugation, but the operation is complex and the cost is high.

[0008] In view of the specificity of Tibetan sheep interstitial cells, it is of great significance to develop an efficient, mild, and high-purity isolation and purification method. This not only helps to deeply study the biological characteristics of Tibetan sheep, but also provides a high-quality cell source for the construction of plateau animal models, disease treatment, and tissue engineering. Therefore, this method aims to optimize the isolation and purification process of primary interstitial cells of Tibetan sheep, improve cell yield, activity, and purity, and provide technical support for related research. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for isolating and purifying primary interstitial cells of Tibetan sheep.

[0010] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0011] The present invention provides a method for isolating and purifying primary interstitial cells of Tibetan sheep, comprising the following steps:

[0012] (1) Mix and digest lamb testis tissue with type IV collagenase and bovine serum albumin, centrifuge and discard the supernatant to obtain an enzymatic hydrolysate;

[0013] (2) Mix and digest the enzymatic hydrolysate with trypsin, filter, centrifuge and discard the supernatant, and obtain a testicular single-cell suspension after washing;

[0014] (3) Centrifuge and discard the supernatant after primary culturing the testicular single-cell suspension, and separate the obtained cell precipitate to obtain primary interstitial cells of the testis.

[0015] Preferably, in step (1), the volume ratio of the lamb testis tissue to type IV collagenase is 1:4-5; the concentration of the type IV collagenase is 0.5-1.5 mg / mL; the final concentration of the bovine serum albumin in the digestion system is 0.4-0.6% (m / v).

[0016] Preferably, in step (1), the digestion time is 0.5-1.5 h, the temperature is 34-38 °C, and it is shaken once every 8-12 min.

[0017] Preferably, in step (2), the volume ratio of the enzymatic hydrolysate to trypsin is 1:2-3; the volume concentration of the trypsin is 0.2-0.3%.

[0018] Preferably, in step (2), the digestion time is 15-25 min, the temperature is 34-38 °C, and it is shaken once every 4-6 min.

[0019] Preferably, in step (2), the filtration is sequentially through cell sieves with 80-120 meshes, 180-220 meshes and 280-320 meshes.

[0020] Preferably, in step (3), the temperature of the primary culturing is 36-38 °C, the carbon dioxide concentration is 4-6%, and the time is 5-7 h.

[0021] Preferably, the separation method in step (3) is: 1) Prepare percoll separation liquids with different concentrations; 2) Add the percoll separation liquids with different concentrations to the separation tube in sequence according to the density for equilibration, resuspend the cell precipitate and add it to the top layer of the equilibrated separation liquid, and after centrifugation, take the suspension between the Percoll gradients with a volume concentration of 34%-60%, and the separation is completed.

[0022] Preferably, the volume concentrations of the percoll separation solution in step 1) are 20-22%, 25-27%, 33-35% and 58-62% respectively.

[0023] The present invention also provides the application of the testicular primary interstitial cells prepared by the described method in the construction of testicular organoids.

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

[0025] The method of the present invention can quickly obtain a large number of high-purity testicular primary interstitial cells, and the method of the present invention is simple, easy to operate, has good repeatability and low cost, and is suitable for large-scale application and promotion. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0027] Figure 1 Is the cell suspension after digestion of testicular tissue.

[0028] Figure 2 Is the cell suspension after the cell suspension is separated by Percoll solution.

[0029] Figure 3 Is the cell morphology of interstitial cells cultured in complete medium for 12 h.

[0030] Figure 4 After culturing testicular primary cells for 24 h, the adherent cells are spindle-shaped or irregularly round.

[0031] Figure 5 After culturing testicular primary cells for 48 h, most of the adherent cells are spindle-shaped.

[0032] Figure 6 After culturing testicular primary cells for 72 h, all the adherent cells are spindle-shaped, and most of them are interstitial cells and Sertoli cells.

[0033] Figure 7 Is the identification of interstitial cells by RT-PCR and immunofluorescence staining; (A) RT-PCR results; (B) Immunofluorescence staining results.

[0034] Figure 8The viability of testicular primary cells separated by different methods: ① 1 mg / mL type I collagenase + 0.25% trypsin; ② 1 mg / mL type IV collagenase + 0.25% trypsin; ③ 1 mg / mL type IV collagenase + 0.5% BSA + 0.25% trypsin.

[0035] Figure 9 The positive cell ratio of interstitial cells separated by different methods: ① 1 mg / mL type I collagenase + 0.25% trypsin; ② 1 mg / mL type IV collagenase + 0.25% trypsin; ③ 1 mg / mL type IV collagenase + 0.5% BSA + 0.25% trypsin. Detailed implementation method

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

[0037] (1) Mix and digest lamb testicular tissue with type IV collagenase and bovine serum albumin, centrifuge and discard the supernatant to obtain an enzymatic hydrolysate;

[0038] (2) Mix and digest the enzymatic hydrolysate with trypsin, filter, centrifuge and discard the supernatant, and wash to obtain a testicular single cell suspension;

[0039] (3) After preliminary culturing the testicular single cell suspension, centrifuge and discard the supernatant, and separate the obtained cell pellet to obtain primary testicular interstitial cells.

[0040] In the present invention, the volume ratio of the lamb testicular tissue to type IV collagenase in step (1) is 1:4 - 5; preferably 1:4.5.

[0041] In the present invention, the final concentration of bovine serum albumin in the digestion system in step (1) is 0.4 - 0.6% (m / v); preferably 0.5% (m / v).

[0042] In the present invention, the concentration of the type IV collagenase is 0.5 - 1.5 mg / mL; preferably 0.7 - 1.3 mg / mL; further preferably 0.9 - 1.1 mg / mL; more preferably 1 mg / mL.

[0043] In the present invention, the digestion time in step (1) is 0.5 - 1.5 h, the temperature is 34 - 38 °C, and shake once every 8 - 12 min; preferably the digestion time is 1 h, the temperature is 35 - 37 °C, and shake once every 9 - 11 min; further preferably the digestion time is 1 h, the temperature is 37 °C, and shake once every 10 min.

[0044] In the present invention, the volume ratio of the enzymatic hydrolysate to trypsin in step (2) is 1:2 - 3; preferably 1:2.5.

[0045] In the present invention, the volume concentration of the trypsin is 0.2-0.3%; preferably 0.25%.

[0046] In the present invention, for the digestion in step (2), the time is 15-25 min, the temperature is 34-38 °C, and it is shaken once every 4-6 min; preferably, the digestion time is 17-23 min, the temperature is 35-37 °C, and it is shaken once every 5 min; more preferably, the digestion time is 20 min, the temperature is 37 °C, and it is shaken once every 5 min.

[0047] In the present invention, for the filtration in step (2), it is sequentially filtered through cell sieves with 80-120 meshes, 180-220 meshes and 280-320 meshes; preferably, the filtration is sequentially through cell sieves with 90-110 meshes, 190-210 meshes and 290-310 meshes; more preferably, the filtration is sequentially through cell sieves with 100 meshes, 200 meshes and 300 meshes.

[0048] In the present invention, for the primary culture in step (3), the temperature is 36-38 °C, the carbon dioxide concentration is 4-6%, and the time is 5-7 h; preferably, the primary culture temperature is 37 °C, the carbon dioxide concentration is 5%, and the time is 6 h.

[0049] In the present invention, the method for separation in step (3) is as follows: 1) Prepare percoll separation liquids with different concentrations; 2) Add the percoll separation liquids with different concentrations to the separation tube in order of density for equilibration. After resuspending the cell precipitate, add it to the top layer of the equilibrated separation liquid. After centrifugation, take the suspension between the Percoll gradients with a volume concentration of 34%-60%, and the separation is completed.

[0050] In the present invention, the volume concentrations of the percoll separation liquids in step 1) are 20-22%, 25-27%, 33-35% and 58-62% respectively.

[0051] The present invention also provides the application of the testicular primary interstitial cells prepared by the above method in the construction of testicular organoids.

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] I. Test materials

[0055] One-month-old Tibetan sheep lambs at birth

[0056] II. Test preparation

[0057] Sterile scissors, forceps, scalpel, laminar flow hood, disposable cell sieves of 100, 200, and 300 meshes (sterile), 75% ethanol, penicillin-streptomycin (gibco, 15140163), PBS buffer (Solarbio, P1020), HBSS buffer (gibco, 14025092), T25 cell culture flask (Corning, CLS430639), 6-well plate (Costar, 3516), 0.5% trypsin (gibco, 15050065), type IV collagenase (sigma, C5138), Percoll cell separation solution (Sigma, P1644), red blood cell lysis buffer (Solarbio, R1010), DMEM / F12 (gibco, 11330032), fetal bovine serum (gibco, 10099-141C).

[0058] III. Reagent Preparation

[0059] ① Type IV collagenase (sigma, C5138)

[0060] Weigh 20 mg of type IV collagenase and dissolve it in 20 mL of calcium- and magnesium-free PBS. After complete dissolution, filter it through a 0.22 μm filter to prepare a 1 mg / mL cell digestion solution;

[0061] Weigh 20 mg of type I collagenase and dissolve it in 20 mL of calcium- and magnesium-free PBS. After complete dissolution, filter it through a 0.22 μm filter to prepare a 1 mg / mL cell digestion solution;

[0062] ② Different gradients of Percoll separation solution (Sigma, P1644):

[0063] a. 21%: Pipette 0.42 mL of Percoll stock solution and add it to 1.58 mL of calcium- and magnesium-free PBS to dilute it to a 21% Percoll solution;

[0064] b. 26%: Pipette 0.52 mL of Percoll stock solution and add it to 1.48 mL of calcium- and magnesium-free PBS to dilute it to a 26% Percoll solution;

[0065] c. 34%: Pipette 0.68 mL of Percoll stock solution and add it to 1.32 mL of calcium- and magnesium-free PBS to dilute it to a 34% Percoll solution;

[0066] d. 60%: Pipette 1.2 mL of Percoll stock solution and add it to 0.8 mL of calcium- and magnesium-free PBS to dilute it to a 60% Percoll solution;

[0067] ③ 0.25% Trypsin working solution

[0068] Absorb 5 mL of 0.5% trypsin solution and add it to 10 mL of calcium- and magnesium-free PBS to dilute it into a 0.25% trypsin working solution;

[0069] ④ Preparation of complete medium: DMEM / F12 medium (gibco, 11330032), 15% FBS (gibco, 10099-141C), 2% penicillin-streptomycin (gibco, 15140163);

[0070] IV. Isolation, purification and culture of primary stromal cells

[0071] It mainly includes the following steps:

[0072] 1. Take lamb testicular tissue and place it in 75% ethanol for rapid disinfection for 1 min;

[0073] 2. In a laminar flow hood, wash it 3 times with PBS containing 1% PS;

[0074] 3. Take sterile scissors, cut off the external tunica albuginea and epididymis of the testis, avoid the external blood vessels of the testis and cut it in half from the middle. Take about 200 mg of tissue blocks from the middle of both testes, transfer the tissue blocks into a culture dish containing HBSS, cut them into 1 mm × 1 mm small pieces with scissors and use the following treatment methods to obtain a cell suspension.

[0075] ① Add 4-5 times the volume of 1 mg / mL type I collagenase to a 50 mL centrifuge tube, transfer the above minced tissue into the centrifuge tube and place it in a 37°C water bath for digestion for 0.5 h, shaking once every 10 min; centrifuge at 800 r / min for 5 minutes and discard the supernatant. Add 3 times the volume of 0.25% trypsin, mix well and place it in a 37°C water bath for digestion for 20 min, shaking once every 5 min. Subsequently, add an equal volume of complete medium to the above 50 mL centrifuge tube to terminate digestion, pipette and mix well, then filter it through 100-mesh, 200-mesh and 300-mesh cell sieves in sequence. After centrifuging the above cell filtrate at 1000 r / min for 5 min, discard the supernatant, then wash the precipitate 3 times with PBS containing 1% double antibody, and resuspend the cell precipitate with complete medium to obtain a single cell suspension a of the testis.

[0076] ② Add 5 times the volume of 1 mg / mL type IV collagenase to a 50 mL centrifuge tube. Transfer the above-mentioned minced tissue into the centrifuge tube and place it in a 37 °C water bath for digestion for 0.5 h, shaking it every 10 min; centrifuge at 800 r / min for 5 minutes and discard the supernatant. Add 3 times the volume of 0.25% trypsin, mix well and place it in a 37 °C water bath for digestion for 20 min, shaking it every 5 min. Subsequently, add an equal volume of complete medium to the above 50 mL centrifuge tube to terminate digestion. Pipette and mix well, then filter it successively through 100-mesh, 200-mesh and 300-mesh cell sieves. Centrifuge the above cell filtrate at 1000 r / min for 5 min and discard the supernatant. Then wash the precipitate 3 times with PBS containing 1% double antibiotics, and resuspend the cell precipitate with complete medium to obtain the single-cell suspension b of the testis.

[0077] ③ Add 5 times the volume of 1 mg / mL type IV collagenase and 2 mL of 0.5% BSA to a 50 mL centrifuge tube. Transfer the above-mentioned minced tissue into the centrifuge tube and place it in a 37 °C water bath for digestion for 0.5 h, shaking it every 10 min; centrifuge at 800 r / min for 5 minutes and discard the supernatant. Add 3 times the volume of 0.25% trypsin, mix well and place it in a 37 °C water bath for digestion for 20 min, shaking it every 5 min. Subsequently, add an equal volume of complete medium to the above 50 mL centrifuge tube to terminate digestion. Pipette and mix well, then filter it successively through 100-mesh, 200-mesh and 300-mesh cell sieves. Centrifuge the above cell filtrate at 1000 r / min for 5 min and discard the supernatant. Then wash the precipitate 3 times with PBS containing 1% double antibiotics, and resuspend the cell precipitate with complete medium to obtain the single-cell suspension c of the testis.

[0078] 4. Purify the cell suspension using the following treatment methods to enrich primary interstitial cells with high purity.

[0079] Take a T25 culture flask and add 5 mL of each of the above-mentioned a, b, and c cell suspensions respectively. Place it in a 37 °C 5% CO2 incubator for culturing for 6 h. Collect the cell suspension, centrifuge at 1000 r / min for 5 min and discard the supernatant, and resuspend the cell precipitate with 1 mL of complete medium. Take 2 mL of each of the prepared different gradient percoll separation liquids, add them to a 15 mL centrifuge tube in order of decreasing density and pre-equilibrate and stabilize them at 4 °C. Gently add 1 mL of the cell suspension to be separated along the inner wall of the centrifuge tube to the top layer of the equilibrated separation liquid. After centrifuging at 3000 r / min for 30 min, take the suspension between the 34% - 60% Percoll gradient;

[0080] 5. Transfer the suspension obtained in step 4 to a new 15 mL centrifuge tube, wash it 2 - 3 times with complete medium, and finally resuspend the cells with complete medium and plate them for culturing to obtain primary interstitial cells of the testis with high purity.

[0081] Experimental Example 1

[0082] Take the single-cell suspension c of the testis obtained in Step 3 of Example 1 and conduct identification as Figure 1 shown, the cells are transparent and round.

[0083] Take the suspension between 34% - 60% Percoll gradient obtained in Step 4 of Example 1 and conduct identification on the cell morphology as Figure 2 shown.

[0084] After culturing the cells obtained in Step (5) of Example 1 for 12 h (37 °C, 5% CO2), the cells start to be oval-shaped ( Figure 3 ), after culturing for 24 h, some cells adhere to the wall and are spindle-shaped ( Figure 4 ). After culturing for 48 h, most of the adherent cells are spindle-shaped, and a small number of transparent and round cells proliferate around the spindle-shaped cells ( Figure 5 ). After culturing for 72 h, all the adherent cells are spindle-shaped, and most of them are interstitial cells and Sertoli cells ( Figure 6 ).

[0085] Experimental Example 2

[0086] Conduct agarose gel electrophoresis and immunofluorescence staining on the interstitial cells obtained in Step (5) of Example 1 (prepared from the single-cell suspension c of the testis). The test method is as follows:

[0087] 1. Reverse transcription polymerase chain reaction

[0088] (1) Collect the cells after adherent proliferation, and extract the total cellular RNA by the Teizol method;

[0089] (2) Synthesize the first-strand cDNA using the Evo M-mLV RT Kit with gDNA Clean for qPCR kit.

[0090] (3) Conduct PCR amplification with a total reaction system of 20 μL (10 μL 2×Pro Taq Master Mix, 1 μL cDNA sample, 0.5 μL upstream primer, 0.5 μL downstream primer, 8 μL RNase-free water). The primer information is shown in Table 1 below.

[0091] Table 1 Primer information and uses

[0092]

[0093] As shown in SEQ ID NO: 1 - 4.

[0094] The amplification reaction procedure is as follows:

[0095] 94°C (pre-denaturation) for 30 s, 1 cycle

[0096] 98°C (denaturation) for 10 s

[0097] 60°C (annealing) for 30 s, 35 cycles

[0098] 72°C (extension) for 1 min

[0099] 72°C (final extension) for 2 min, 1 cycle

[0100] (4) Take 5 μL of the amplified product and add it to a 2% agarose gel for RT-PCR;

[0101] (5) Observe the positions and sizes of the marker, GAPDH, and HSD3B1 bands in a Gel Imaging Systems.

[0102] 2. Immunofluorescence staining

[0103] (1) Use a PAP PEN immunohistochemical pen to draw a circle on an adhesive-free glass slide, drop the interstitial cell suspension into each circle, and bake it in an oven at 60°C for 30 min;

[0104] (2) Drop 4% paraformaldehyde to fix for 15 min;

[0105] (3) Wash with PBS 3 times, 5 min each time;

[0106] (4) Drop 0.3% TritonX-100 for permeabilization for 10 min;

[0107] (5) Wash with PBS 3 times, 5 min each time;

[0108] (6) Drop the blocking solution and block at room temperature for 60 min;

[0109] (7) Drop the primary antibody HSD3B1 (1:500) and incubate overnight at 4°C;

[0110] (8) Wash with PBST 3 times, 5 min each time;

[0111] (9) Drop the goat anti-rabbit secondary antibody (1:200) and incubate for 1 h at room temperature in the dark;

[0112] (10) Wash with PBST 3 times, 5 min each time;

[0113] (11) Drop the DAPI solution for nuclear staining and incubate for 10 min at room temperature in the dark;

[0114] (12) Wash with PBST 3 times, 5 min each time;

[0115] (13) Observe the cells under an inverted fluorescence microscope.

[0116] The test results are as Figure 7 shown. The purified interstitial cells were subjected to agarose gel electrophoresis and immunofluorescence staining. The results of agarose gel electrophoresis showed that the purified primary interstitial cells highly expressed HSD3B1. Therefore, the purified cells were interstitial cells. Immunofluorescence staining was performed using the interstitial cell-specific marker antibody HSD3B1. The results showed that the HSD3B1 protein had strong fluorescence signals and was mainly localized in the cytoplasm. Therefore, the results of agarose gel electrophoresis and immunofluorescence staining further proved that the purified cells were interstitial cells.

[0117] 3. Detection of the proliferation of primary testicular interstitial cells

[0118] The Cell Counting Kit-8 (CCK-8, ZETALIFE, C7661) was used to detect cell proliferation. The primary cells obtained from a, b, and c above were seeded into 96-well plates at a density of 2×10 3 . 10 μL of CCK-8 was added to each well and incubated at 37 °C in 5% CO2 for 2 h. Then, the absorbance was measured at 450 nm using a Multiskan FC microplate reader. Each group had 6 replicates.

[0119] It can be seen from Figure 8 and Figure 9 that three two-step enzyme digestion methods were used to isolate primary testicular cells, and 3β-HSD staining was used for staining. The results showed that method ③ had a better effect on isolating interstitial cells.

[0120] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating and purifying primary interstitial cells of Tibetan sheep, characterized in that, It includes the following steps: (1) Mix and digest lamb testis tissue with type IV collagenase and bovine serum albumin, centrifuge and discard the supernatant to obtain an enzymatic hydrolysate; (2) Mix and digest the enzymatic hydrolysate with trypsin, filter, centrifuge and discard the supernatant, and wash to obtain a testicular single cell suspension; (3) After primary culturing the testicular single cell suspension, centrifuge and discard the supernatant. After separating the obtained cell pellet, primary testicular interstitial cells are obtained.

2. The method according to claim 1, wherein In step (1), the volume ratio of the lamb testis tissue to type IV collagenase is 1:4 - 5; the concentration of the type IV collagenase is 0.5 - 1.5 mg / mL; the final concentration of the bovine serum albumin in the digestion system is 0.4 - 0.6% (m / v).

3. The method according to claim 1, wherein In step (1), the digestion time is 0.5 - 1.5 h, the temperature is 34 - 38 °C, and it is shaken every 8 - 12 min.

4. The method according to claim 1, wherein In step (2), the volume ratio of the enzymatic hydrolysate to trypsin is 1:2 - 3; the volume concentration of the trypsin is 0.2 - 0.3%.

5. The method according to claim 1, wherein In step (2), the digestion time is 15 - 25 min, the temperature is 34 - 38 °C, and it is shaken every 4 - 6 min.

6. The method according to claim 1, characterized in that, The filtration in step (2) is sequentially through cell sieves with 80 - 120 meshes, 180 - 220 meshes, and 280 - 320 meshes.

7. The method according to claim 1, characterized in that, In step (3), the temperature of the primary culture is 36 - 38 °C, the carbon dioxide concentration is 4 - 6%, and the time is 5 - 7 h.

8. The method according to claim 1, characterized in that The separation method in step (3) is as follows: 1) Prepare percoll separation liquids with different concentrations; 2) Add the percoll separation liquids with different concentrations to the separation tube in order of density for equilibration. After resuspending the cell pellet, add it to the top layer of the equilibrated separation liquid. After centrifugation, take the suspension between the Percoll gradients with a volume concentration of 34% - 60%, and the separation is completed.

9. The method according to claim 8, wherein In step 1), the volume concentrations of the percoll separation liquids are 20 - 22%, 25 - 27%, 33 - 35%, and 58 - 62% respectively.

10. Application of the primary testicular interstitial cells prepared by the method according to any one of claims 1 - 9 in the construction of testicular organoids.

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