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.