Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

1. Animal Ethics Statement

Experiments described here were performed according to the guidelines of the Regierungspräsidium Giessen, Germany, as the local authority and confirm to the German Code of Practice for the Care and Use of Animals for Experimental Purposes (Permission no: GI 20/23 Nr. A 31/2012).

2. Preparation of Media, Enzyme Solutions and Animals

1. Prepare 1% iodine alcohol by dissolving 1 g of iodine in 100 ml ethanol.
2. Prepare 2x 500 ml Dulbecco´s phosphate buffered saline (PBS) without Ca²⁺ and Mg²⁺ supplemented each with 7.5 ml D-glucose (100 g/L) and 5 ml 100x Penicillin/Streptomycin stock solution (15 mg/ml D-glucose, 50 U/ml Penicillin and 50 µg/ml Streptomycin final).
3. Supplement 1x 500 ml Roswell Park Memorial Institute (RPMI) 1640 medium with 5 ml 100x Penicillin/Streptomycin stock solution (50 U/ml Penicillin and 50 µg/ml Streptomycin final).
4. Prepare a Trypsin-DNase I solution (2.5 mg/ml Trypsin and 10 µg/ml DNase I) by adding 25 mg Trypsin and 0.1 ml DNase I (1 mg/ml DNase I) to 9.9 ml PBS.
5. Dissolve 50 mg Trypsin inhibitor in 5 ml PBS for Trypsin inhibitor A solution (10 mg/ml). Dissolve 25 mg Trypsin inhibitor in 10 ml PBS for Trypsin inhibitor B solution (2.5 mg/ml).
6. For a Collagenase-Hyaluronidase-Deoxyribonuclease I (DNase I) solution dissolve 10 mg Collagenase A (1 mg/ml final), 10 mg Hyaluronidase (1 mg/ml final) and 0.1 ml DNase I (1 mg/ml DNase I) (10 µg/ml final) in 10 ml PBS.
7. Prepare a Hyaluronidase-DNase I solution by adding 10 mg Hyaluronidase (1 mg/ml final) and 0.1 ml DNase I solution (1 mg/ml) (10 µg/ml final) to 9.9 ml PBS.
8. Order Wistar rats on time so that they are 19 days old on the day of Sertoli cell isolation.
   Note: The described protocol is designed for 10 rats but can be modified for a minimum of 5 and a maximum of 20 rats. The volumes of all solutions have to be adjusted accordingly.
9. Prepare Oil Red O stock solution by dissolving 0.35 g Oil Red O in 100 ml isopropanol. Stir O/N, filter through 0.2 µm and store at RT for up to one year.
10. Prepare Oil Red O staining solution by mixing 6 ml Oil Red O stock solution with 4 ml water (3:2). After 10-20 min filter through 0.2 µm. Use within the next 2 hr.
11. Prepare a 4% (w/v) formaldehyde solution in a ventilated hood by adding 4 g paraformaldehyde powder (PFA) to 80 ml of 1x PBS while stirring gently. Heat to ~60 °C, add 1 ml of 1 M NaOH and continue stirring until the paraformaldehyde is dissolved. Let the solution cool down to RT, adjust the pH to 7.4 with 1 M HCl and the volume to 100 ml with 1x PBS. Filter the solution (0.45 µm) and use fresh or store in aliquots at −20 °C for several months.
    Note: Polymerized formaldehyde (PFA) depolymerizes to monomeric formaldehyde in water. This process is catalyzed by moderate heating and a slightly alkaline pH. Prepare all enzyme solutions freshly and filter sterilize (0.2 µM) them on the day of use. If a different manufacturer for an enzyme (see Materials/Equipment Table) is used, carefully adjust its concentration/activity.
    In the following protocol a "pipette" stands for a serological pipette.

3. Preparation of Seminiferous Tubules

1. Anesthetize 10 male Wistar rats one by one in a desiccator using CO₂ and a flow rate that displaces at least 20% of the chamber volume per minute. Wait until breathing stops, test anesthesia by squeezing a foot pad, and if there is no reaction euthanize each rat by cervical dislocation. Drain the blood by sectioning jugular vein and carotid artery (vagina carotica) under flowing water. Disinfect the abdomen by wiping with 70% ethanol.
2. Using forceps lift the skin from the abdominal muscles and cut out an oval shaped skin lobe that is extending from the pubic symphysis to the sternum (Figure 2A) and flap it upwards onto the chest. Next, grab the abdominal muscles and make a medial incision from the pubic symphysis to the sternum.
   1. Squeeze the abdomen with the thumbs from pelvis upwards in order to push the testes out of the lower pelvis. Pick the epididymal fat pad (Figure 2B) with forceps for pulling up the testis further. Cut the spermatic cord (Figure 2B), leaving the tunica albuginea intact, and collect all testes in 20 ml PBS in a 50 ml conical tube (Figure 2C).
3. After collecting all testes, disinfect them by adding an equal volume (20 ml) of 1% (w/v) iodine in ethanol. Invert the tube twice and decant the supernatant immediately. Quickly wash the testes twice with 25 ml PBS each (Figure 2D).
4. Transfer the testes to a Petri dish containing 15 ml PBS (Figure 2E). Grasp each testis firmly by forceps at one end, make a small incision into the tunica albuginea at the opposite end, and squeeze out the tubules using closed scissor blades as a scraping tool.
   Note: The tubules should still form a compact testicle-shaped bundle with only a few tubules extending into the solution in order to enable homogeneous enzymatic digestion (Figure 2F).
5. Transfer the de-capsulated testes to a 100 ml screw-cap bottle containing 10 ml Trypsin-DNase I solution. Perform the digestion in a shaking water bath at 32 °C (120 oscillations/min). After 4 min evaluate the tubules with the naked eye for dispersion of tubules. Once tubules start to disperse into the solution, stop the digestion immediately. If necessary, continue incubation for up to 2 min.
6. Stop trypsin digestion by adding 5 ml of trypsin inhibitor solution A. Thoroughly mix the solution by pipetting up and down 3-4 times using a 10 ml pipette and transfer it to a 50 ml conical tube.
7. After 5 min of incubation observe the tubules settle by unit gravity, carefully remove the supernatant by using the 10 ml pipette from step 3.6 and add 10 ml of trypsin inhibitor B solution in order to fully stop trypsin digestion. Resuspend the tubules by pipetting up and down 2-3 times.
8. In order to remove contaminating interstitial cells wash the tubules 9 times with 25 ml PBS each. Resuspend tubules in each wash by using a 25 ml pipette and allow them to settle by unit gravity for 10 min. Always use the same 25 ml pipette for pipetting PBS, resuspension and removal of the supernatant.

4. Removal/Isolation of Peritubular Cells (PTCs)

1. Transfer all tubules to a 100 ml screw-cap bottle and add the Collagenase-Hyaluronidase-DNase I solution (Figure 3A). Perform digestion for 10 min in a shaking water bath at 32 °C (120 oscillations/min).
2. Pipet a drop of the suspension on a glass slide and quickly check the tubules under an inverted light microscope for routine cell culture at 100x magnification. If digestion is not advanced enough continue incubation for additional 2 min (do not count the time needed for the microscopical check).
   Note: During this step all tubules get shortened in length and tubule edges should become rough (Figure 3B and C). Rough edges are indicative for the release of peritubular cells.
3. Transfer the suspension with digested tubules to a 50 ml conical tube using the 25 ml pipette from step 3.8. Wash the 100 ml bottle with 10 ml PBS and add it to the tubules in the conical tube. Allow the digested tubules to settle by unit gravity for 10 min, and carefully aspirate the supernatant, containing the PTCs, using the 25 ml pipette again, and transfer it to a new conical tube. For aspiration of the last few milliliters use a 5 ml pipette in order to prevent any carry-over of tubules.
4. Add 20 ml RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) to the collected tubule supernatants, mix and centrifuge at 300 x g for 10 min at RT without using the break.
5. Carefully decant the supernatant and resuspend the pelleted PTCs in 20 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS and seed 4 ml each on five T75 flasks containing 16 ml medium each. Incubate at 37 °C in 5% CO₂ atmosphere (Figure 4A).
6. On the 3^rd day of culture trypsinize the PTCs and split them 1:2 on T75 flasks containing 16 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS each (yields 10 flasks altogether) (Figure 4B). Continue incubation at 37 °C in 5% CO₂ atmosphere.
7. On the 5^th day of culture trypsinize the PTCs again and plate them on 6-well or 24-well plates depending on the experiment (one T75 flask per plate). Experiments can be performed on day 6.

5. Isolation of Sertoli Cells (SCs)

1. Resuspend the settled tubules from step 4.3 thoroughly in 25 ml of PBS by using a 25 ml pipette and allow them to settle by unit gravity for 12 min. Use the same 25 ml pipette for pipetting PBS, resuspension of tubules and removal of supernatants. Repeat the wash 3 times (4 washes in total).
2. After the last wash transfer the digested tubules to another 100 ml screw-cap bottle containing the Hyaluronidase-DNase I solution. Perform the digestion in a shaking water bath at 32 °C (120 oscillations/min).
3. After 5 min check the tubules again by light microscopic inspection at 100X magnification as described in step 4.2.
   Note: Short tubules, tubular aggregates and released cells should be visible (Figure 3D).
   1. Allow the tubular aggregates settle for 10 min, and aspirate the supernatant using a 25 ml pipette. Wash aggregates 4 times using 25 ml PBS and a sedimentation time of 10 min for each washing step (Figure 3E).
4. Add 20 ml of RPMI 1640 medium and pass the suspension 10 times through an 18 G needle mounted on a 20 ml syringe. Avoid air bubbles.
   Note: Pulling the suspension in and out through the needle is considered as one pass. The disruption and homogenization of cells by hydrodynamic shearing is a standard technique in the preparation of cells that tend to aggregate. The passing of cells through a hypodermic needle of small inner diameter is unlikely to have an impact on their functional properties²⁵. Figure 5C and D shows the normal ultrastructure of purified Sertoli cells on day 4 of culture.
   1. Pipet a drop of the suspension on a glass slide and quickly check the tubules under an inverted light microscope for routine cell culture at 10X magnification if the aggregates are all dispersed (Figure 3F). Filter the cell suspension through a 70 µm cell strainer in order to obtain a pure single cell suspension in the filtrate.
5. Centrifuge the filtrate of step 5.4 at 200 x g for 10 min at RT without using the break. Carefully aspirate the supernatant using a 25 ml pipette, and resuspend the Sertoli cells in 40 ml RPMI 1640 medium (serum-free).
6. Mix 100 µl cell suspension with 100 µl of Trypan Blue Stain and count cells with a Neubauer Improved chamber. Adjust the cell concentration to 3 x 10⁶ cells/ml according to the counting result. Seed 1 ml per well on a 6-well plate (=day 1). If an Oil Red O staining (step 6) is planned put a cover slip into one or a few wells before seeding Sertoli cells.
   Note: Sertoli cells in suspension settle quickly so that the tube should be briefly shaken every time before a cell aliquot is taken with the pipette. Until day 4 they firmly attach to the substratum.
7. In order to remove floating or adherent germ cells on day 4 (Figure 4C) wash each well of the 6-well plates used for plating isolated Sertoli cells (step 5.6) with PBS (Figure 4D). Aspirate the medium, add 1-2 ml of PBS to each well and shake the 6-well plates horizontally on a table vigorously but without spilling PBS. Repeat the wash 3 times and perform the same washing procedure on day 5 and 6 (Figure 4E).
   Note: On day 4 Sertoli cells have firmly attached and show a cobblestone like pattern under the inverted light microscope (Figure 4C). Experiments can be performed from day 7 onwards.
   1. (Alternative for step 5.7) For the removal of floating and adherent germ cells perform a hypotonic shock 3 days after seeding on 6-well plates. Remove the medium and add 1 ml of 20 mM Tris pH 7.5 taken directly out of the fridge. Aspirate the Tris buffer after 90 to 120 sec but not later. Wash cells twice with PBS, and add 2 ml of RPMI 1640 medium (serum-free). Optionally, perform experiments the next day.
      Note: The longer the duration of the hypotonic shock is the more germ cells can be removed but the more Sertoli cells get lost as well. The first PBS wash should be performed quickly but with care in order not to displace any Sertoli cells. Directly after hypotonic treatment numerous vacuoles of different sizes can be observed in the cytoplasm by phase contrast microscopy. Vacuoles disappear within a few hours after hypotonic treatment.

6. Oil Red O Staining

1. Perform all steps at RT. On day 7 wash Sertoli cells grown on a cover slip (step 5.6) with PBS twice and fix them with 10% formalin in PBS. After 10 min replace formalin with fresh solution, and continue fixation for another 60 min.
   Note: All solutions are added to the well(s) with a cover slip and aspirated before the next step.
2. Aspirate formalin, wash cells with water twice and add 60% isopropanol. After 5 min allow cells to air dry for a few minutes.
3. Add 1 ml Oil Red O staining solution per well and incubate for 10 min. Aspirate the solution, and wash cells immediately 4 times with water and mount cover slips in glycerol-PBS (9:1). Take bright field images with a routine light microscope (Figure 4F).

7. Immunofluorescence Staining

1. Wash PTCs (from step 4.5) or Sertoli cells (from step 5.5) grown on a cover slip with PBS twice and fix them with 4% PFA for 10 min at RT.
2. Incubate with 5% BSA in PBS for 1 hr at RT. Discard the BSA-PBS solution and add fresh BSA-PBS solution containing actin (smooth muscle) (for PTCs) or vimentin (for Sertoli cells) primary antibody (dilution 1:100 for both antibodies) and incubate at 4 °C O/N.
   Note: Incubation with BSA blocks non-specific binding of the primary antibody.
3. Wash cover slips 3 times for  5 min in PBS-0.1% Tween 20 and incubate with green-fluorescent dye conjugated goat anti-mouse secondary antibody (dilution 1:1,000) at RT for 1 hr in the dark.
4. Wash cover slips 3 times for 5 min in PBS-0.1% Tween 20 and mount them in DAPI mounting medium.

8. Ultrastructural Analysis

1. Wash Sertoli cells (from step 5.6) grown on a 6 cm cell culture plate with PBS and fix them for 2 hr at 4 °C in a mixture of 2.5% glutaraldehyde, 2.5% paraformaldehyde and 0.05% picric acid in 67 mM cacodylate buffer (pH 7.4) according to Ito and Karnovsky²⁶.
2. Perform post-fixation in 1% osmium tetroxide followed by an O/N incubation in 0.3% uranyl acetate dissolved in 50 mM maleate buffer (pH 5). Embed samples in embedding media according to standard procedures. Cut thin sections, contrast them with lead citrate and examine with an electron microscope.