Abstract
Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male’s lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.
Highlights
Spermatogonial stem cells (SSCs) are adult germline stem cells that serve as the foundation of spermatogenesis throughout the lifetime of a male
The objective of the present study was to evaluate the effectiveness of various cryopreservation media additives, including antioxidants and apoptosis inhibitors, on the recovery, proliferation capacity, and mitochondrial activity of thawed germ cells enriched for SSCs
Following cryopreservation for 1 month in various cryopreservation media, thawed cells were evaluated for recovery rate, proliferation capacity, and mitochondrial activity
Summary
Spermatogonial stem cells (SSCs) are adult germline stem cells that serve as the foundation of spermatogenesis throughout the lifetime of a male. Previous work has suggested that SSCs can be cryopreserved [2, 5,6,7,8,9] and that a combination of slow freezing followed by rapid thawing is an effective way to preserve these valuable cells [9, 10]. This method of preservation, as opposed to long-term culture or germline maintenance via xenotransplantation, may be clinically useful for humans because of its convenience. Previous studies have demonstrated that dimethyl sulfoxide (DMSO) was the most effective PCA for murine SSCs [13]
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