Advancing recovery and cryopreservation of rat spermatogonia for germ stem cell banking

Abstract

Optimisation of spermatogonial cryopreservation medium and spermatogonia recovery procedures essential for the male germ cell research and spermatogonia mediated transgenesis in rodents. Methods. PCR, LIVE/DEAD® Cell Viability Assays, Spermatogonial Colony Forming Assay, immunocytochemistry, cryopreservation and thawing spermatogonia cells. Results. Three Sleeping Beauty mutant spermatogonia stem cell lines were stored in liquid nitrogen, after 17-24 months of cryopreservation, were derived in parallel from recovered cryopreserved cultures via an established laminin selection procedure and by an alternative method termed as direct SG (Spermatogonia Growth) medium selection on MEFs (mouse embrionic fibroblasts). A spermatogenic potential of the cell lines derived by these two methods was verified by the spermatogonia transplantation in vivo and reestablishing the mutant animal lines. We optimized the concentration of DMSO in a freezing medium for spermatogonial cell lines. Conclusions. We developed and optimized recovery of spermatogonial stem cells, that could be consistently derived from the freshly thawed stocks of testicular cells cryopreserved in SG medium. Our data suggest that the 8 % DMSO is the most effective concentration of the cryoprotectant in SG medium.

Inducible dominant negative ErbB2 rat spermatogonial line for generation of transgenic rat model and dissecting ERBB2 tyrosine kinase mediated pathways

“…This procedure was used to isolate a parental transgenic rat spermatogonial line, GCS-EGFP/DAZL-ERT2CreERT2, which stably expresses an inducible form of CRE recombinase (iCRE) selectively in the germline of tgGCS-EGFP rats [38]. After selection on gelatin freshly isolated laminin-binding germ cell were harvested and then plated on irradiated DR4 Mouse Embryonic Fibroblasts (Bio Pioneer Inc.) in duplicate wells (~1.9 cm 2 ) to initiate the subculture of spermatogonial lines [40,41]. Alternatively, cells from the cryopreserved population of the corresponding cell line were plated directly on DR4 Mouse Embryonic Fibroblasts [42,43].…”