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All animal procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat‑sen University (Approval No. SYSU-IACUC-2026-B1278) and were performed in accordance with the institutional guidelines.
1. Isolation and culture of primary mouse SLCs
- Dissect testes from C57BL/6 mice and carefully remove the tunica albuginea.
- Mince the testes into small pieces.
- Dissociate interstitial cells from seminiferous tubules by incubating in 1 mg/mL collagenase type IV in DMEM/F12 at 37 ºC for 15 min.
- Add DMEM/F12 containing 10% fetal bovine serum to stop collagenase activity.
- Centrifuge at 1500 × g for 3 min at room temperature.
- Resuspend the pellet in PBS and filter through a 70 µm filter.
- Enrich CD51⁺ cells by fluorescence-activated cell sorting (FACS) using an Influx Cell Sorter.
- Seed the enriched cells in SLC culture medium composed of DMEM/F12 supplemented with: 1 nM dexamethasone, 1 ng/mL LIF, 5 µg/L insulin‑transferrin‑sodium selenite, 5% chicken embryo extract, 0.1 mM β‑mercaptoethanol, 1% nonessential amino acids, 1% N2, 2% B27, 20 ng/mL basic fibroblast growth factor, epidermal growth factor, platelet‑derived growth factor, and oncostatin M.
- Culture the SLCs under standard conditions (37 °C, 5% CO₂) until they reach the desired density for seeding onto hydrogels.
2. Assembling the gel casting glass container
- Carefully inspect and clean the specialized glass plates and spacers used for casting polyacrylamide gel electrophoresis (PAGE) gels, ensuring they are free of dust, damage, and residues.
- Correctly assemble the glass plates, spacers, and clamps according to the manufacturer's instructions, ensuring the assembly is well-sealed to prevent leakage during gel casting.
- Place the assembled glass container securely on a laboratory stand, ready for casting.
NOTE: The casting setup uses standard Western blot (sodium dodecyl sulfate [SDS]-PAGE) glass plates with fixed spacers of either 1.0 mm thickness, ensuring reproducible gel thickness and a flat surface for cell culture.
3. Preparing PAGE gel mix solution
- Precisely measure the following reagents according to the experimental design (see Table 1) into a 15 mL or 50 mL sterile centrifuge tube: 40% Acrylamide stock solution, 2% Bis-acrylamide stock solution, 1M HEPES buffer (pH 8.5), Sterile ddH₂O.
CAUTION: Acrylamide and bis‑acrylamide are neurotoxins. Always wear gloves and a dust mask when weighing dry powder to avoid skin contact and inhalation of airborne particles. Procedures should be performed within a fume hood.
4. Adding initiator and catalyst, and casting the gel
- Add the following to the mixed solution sequentially: 10% Ammonium Persulfate (APS): Acts as the initiator to start the polymerization reaction. N,N,N',N'-Tetramethylethylenediamine (TEMED): Acts as the catalyst to accelerate the polymerization reaction. For a total gel volume of 6 mL, add 60 µL of 10% APS and 6 µL of TEMED (see Table 1 for scaling).
NOTE: The polymerization reaction begins immediately after adding TEMED; therefore, subsequent steps must be performed swiftly.
5. Pouring the mixed solution
- Immediately pipette or pour the mixed solution steadily along one edge of the glass container to avoid creating bubbles.
6. Allowing the gel to polymerize
- Let the cast gel container sit undisturbed at room temperature.
NOTE: Polymerization usually takes 15–30 min at room temperature (20–25 °C).
- Proceed to the next step once a clear interface between the unpolymerized gel mixture and the polymerized gel is visible and the gel is completely solid (the gel does not flow when the container is tilted).
- Immediately after casting, carefully overlay a thin layer of absolute ethanol (or water-saturated ethanol) on top of the gel mixture to exclude atmospheric oxygen, which inhibits radical polymerization. The ethanol also helps form a flat upper surface.
- After polymerization, remove the ethanol before disassembling the plates.
7. Disassembling the container and harvesting the gel
- Carefully disassemble the clamps and use a plastic wedge or spatula to gently pry the glass plates apart, allowing the polymerized PAGE gel to remain intact on one plate.
- Prepare a culture dish containing an ample amount of sterile PBS.
- Carefully peel the gel from the glass plate using a spatula or gloved hand, and let it slide smoothly into the PBS.
- Seal the dish containing the gel in PBS and incubate at 4 °C overnight.
NOTE: This step is crucial. The newly prepared gel is dehydrated and must fully absorb water and swell to its final dimensions in PBS. If this step is skipped and the gel is cut directly, it will continue to expand in subsequent cell culture medium, likely exceeding the area of the culture well and rendering it unusable. The gel thickness after swelling is 1 mm. This is critical because when the gel thickness falls below 100 µm, cells may sense the stiffness of the underlying rigid culture plate, which would interfere with the intended mechanobiological measurements.
8. Cutting and transferring the gel to a culture plate
- The next day, retrieve the gel from 4 °C. Using a sterile biopsy punch or blade cutter (with a diameter matching the well size of the culture plate, e.g., 6-well plate), cut the gel under sterile conditions.
- Within a biosafety cabinet, use sterile forceps to gently pick up the cut gel disc and place it in the center of an empty, sterile culture well.
9. Initial washing and UV irradiation
- Add sufficient sterile PBS to the well containing the gel, ensuring it is completely submerged.
- Gently rock the culture plate to wash the gel surface.
- Aspirate the PBS using a pipette.
- Repeat this washing process once more, for a total of two washes, each lasting 3 min.
- After washing, add sufficient sterile PBS to cover the gel again. Place the entire culture plate with the lid slightly ajar under a UV lamp (inside a biosafety cabinet or a dedicated UV crosslinker) and irradiate for 1 h for basic sterilization.
10. Activation of Sulfo-SANPAH crosslinker
- Inside the biosafety cabinet, carefully aspirate the PBS from the well.
- Before aspirating, gently separate the gel from the well walls and bottom using a pipette tip to avoid damaging the gel due to liquid surface tension.
- Transfer the gel to another clean well in the same culture plate using sterile forceps.
NOTE: When transferring the gel, first adding a small amount of PBS to the clean well can help expel air bubbles trapped between the well bottom and the gel upon placement, and also serves as an additional wash.
- Prepare crosslinker working solution: Freshly prepare a 0.2 mg/mL Sulfo-SANPAH working solution by diluting the stock solution (see Table 1) using sterile water or the recommended buffer (e.g., PBS).
NOTE: Protect the solution from light.
- Add sufficient Sulfo‑SANPAH working solution to cover the gel (e.g., 250 µL for a 12‑well plate, 500 µL for a 6‑well plate), ensuring the gel is completely covered.
- Place the plate under 365 nm wavelength /120 mJ/cm2 UV light and irradiate for 1 h.
NOTE: This step activates Sulfo-SANPAH, enabling it to bind to functional groups on the gel surface.
11. Crosslinker activation and final washes
- After irradiation, aspirate the Sulfo-SANPAH solution from the well inside the biosafety cabinet.
- Add sufficient sterile PBS to the well, gently rock to wash the gel surface, and aspirate the PBS.
- Repeat this washing process once more, for a total of two washes, each lasting 3 min, to thoroughly remove any unreacted Sulfo-SANPAH.
12. Preparing rat tail type I collagen working solution
- Use a 0.02 M acetic acid solution as a solvent to dilute the rat tail type I collagen stock solution.
- Adjust the final working solution concentration to 60–100 µg/mL. For example, if the collagen stock concentration is 4 mg/mL, add 15–25 µL of stock to 985–975 µL of 0.02 M acetic acid to obtain 1 mL of working solution.
13. Collagen coating and incubation
- Add the appropriate volume of collagen working solution to each well containing the activated gel according to the plate type: add 2 mL for a 6-well plate, or 1 mL for a 12-well plate.
- Gently swirl the culture plate to ensure the collagen solution evenly covers the gel and the well bottom.
- Cover the plate and incubate overnight in a 37 °C, 5% CO₂ incubator to allow the collagen to fix onto the gel surface via Sulfo-SANPAH crosslinking.
14. Secondary UV irradiation and fixation
- The next day, remove the plate and irradiate again under about 120 mJ/cm2 UV light for 1 h.
15. Remove unbound collagen and seed cells
- Inside the biosafety cabinet, carefully aspirate the rat tail type I collagen working solution from the wells.
- Add sterile PBS to the wells, gently rock to wash the gel surface, and aspirate the PBS.
- Repeat the wash once more, for a total of two washes, each lasting 3 min.
- After washing, the gel matrix is ready. Resuspend the prepared stem Leydig cells in the complete SLC culture medium (composition detailed in step 1).
- Seed the cell suspension directly onto the gel surface. The recommended seeding density is 2 × 104 cells/cm2 (e.g., approximately 2 × 105 cells per well for a standard 6-well plate).
- Incubate the culture plates at 37 °C in a humidified 5% CO₂ incubator.
- To maintain optimal cell viability and steroidogenic function, change the culture medium every 48 h.