An effective method for recovering rat spermatogonial stem cells from frozen rat testis cells

Cryopreservation of testis tissue, including spermatogonial stem cells (SSCs), underpins a potential method of fertility preservation for prepubertal males undergoing gonadotoxic treatments. A key understudied factor in the recovery of high viability testis cell populations from frozen is the method of thawing and purification of SSCs. Here, we thawed rat testis cells, testing the effect of gradual dilution with DMSO, supplementation with sugars and removal of dead cells on thawing efficiency. Gradient reduction of DMSO and trehalose supplementation increased thawing viability significantly. Magnetic-activated cell sorting of EpCAM⁺ cells enriched rat SSCs with a concentration superior to that achieved with laminin selection. A combination of methods to remove dead cells and enrich rat SSCs from post thaw rat testis cells provided an optimized protocol. Taken together, we have established a reliable protocol to enrich high-viability SSCs from post thaw rat testis cells, which may serve as a template for purifying SSCs from frozen human testis cells.