Isolation of Immune Regulatory Sertoli Cells from Mouse Seminiferous Tubules

The seminiferous tubules of the testes contain Sertoli cells, which nourish germ cells. Sertoli cells are connected via tight junctions and are firmly attached to the hyaluronic acid-rich basement matrix.  This arrangement of Sertoli cells regulates immune cell entry into the tubule and protects developing germ cells from degradation.

To isolate Sertoli cells, begin with a tube containing partially digested seminiferous tubules derived from mouse testes. These tubules lack peritubular cells — smooth muscle cells surrounding the seminiferous tubules.

Treat the tubules with an enzyme cocktail containing hyaluronidase — essential for the hydrolysis of hyaluronic acid — and DNase.

Hyaluronidases enter the tubules and cleave hyaluronic acid — a basement polysaccharide — initiating Sertoli cell detachment from the basement matrix and releasing germ cells into the solution. DNase digests any DNA contaminants from the solution, preventing cell aggregation.

Wash the enzymatically digested tubules with a buffer, removing residual enzymes, cellular fragments, and germ cells. Resuspend the digested tubules and load the suspension into a needle-syringe assembly.

Pass the solution repeatedly to create hydrodynamic shearing and disrupt the Sertoli cells' tight junctions, releasing them as single cells along with the remnant germ cells.

Filter the cell suspension, removing cellular debris and obtaining a single-cell suspension. Centrifuge the filtrate to pelletize the cells.

Resuspend the cells in a serum-free growth medium for selective expansion of Sertoli cells.

To isolate the Sertoli cells, use a new 25-milliliter pipette to resuspend the settled tubules thoroughly in 25 milliliters of PBS. After allowing the tubules to settle for another 12 minutes, use the same pipette to wash the tubules three more times in the same way. Then, transfer the tubules into a new 100-milliliter screw-cap bottle and add the Hyaluronidase-DNase-I solution. Further, digest the tubules in a shaking water bath at 32 Celsius for five minutes.

Check an aliquot of the suspension by light microscope inspection at 100x magnification. Short tubules, tubular aggregates, and released cells should be visible. Allow the tubular aggregates to settle for 10 minutes. Then, use a new 25-milliliter pipette to remove the supernatant and wash the aggregates four times in 25ml of PBS, with 10 minutes of sedimentation between each wash.

The contaminating PTC population will have been reduced. After the last wash, add 20ml of RPMI 1640 medium to the cells, and pass the suspension through an 18G needle mounted on a 20-milliliter syringe 10 times. Next, quickly confirm that all of the aggregates have been dissociated, and filter the cell suspension through a 70-micron cell strainer to obtain a pure single-cell suspension.

Spin down the filtrate, using the 25-milliliter pipette to carefully aspirate the supernatant. Then, resuspend the isolated Sertoli cells in 40ml of serum-free RPMI 1640 medium. Then, count the number of viable cells by trypan-blue exclusion and adjust the cell concentration to 3 x 10⁶ cells per milliliter. Finally, feed 1 milliliter of cells per well in a six-well plate.