Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells

Abstract

The purpose of this research was to investigate the trehalose and vitamin C (Vit C) co-supplementation of freezing media to create a successful cryopreservation protocol for conservation of ovine spermatogonial stem cells (SSCs). SSCs were cryopreserved and cultured with an essential freezing medium containing 200 mM trehalose, 40 µg/mL Vit C, and a combination of both for 3 weeks. Cell viability, colony number and diameter and mRNA levels of Bax, and Bcl-2 genes were evaluated before and after cryopreservation with quantitative real-time PCR. The results showed that cells cryopreserved in freezing medium containing 200 mM of trehalose plus 40 µg/mL Vit C had considerably greater cell viability than the control group (P<0.0001). Up to the 3rd week of cell culture, supplementation of freezing medium with 200 mM trehalose resulted in significantly lower colonies diameters than in the control group. No significant differences were observed during the 1st to 2nd weeks in colony diameter and number of colonies between cells cryopreserved in the freezing medium containing either Vit C or trehalose compared with the control groups. Following thawing, the mRNA level of Bax in the Vit C + trehalose group was drastically lower than in those treated with trehalose or Vit C only (P<0.0001). High expression of Bcl-2 in the 40 µg/mL Vit C group was recorded in the thawed cells compared to the control group (P<0.0001). These findings indicate that the concomitant use of antioxidants and sugar in the freezing medium can improve the quality and viabi­lity of SSCs after freezing via different mechanisms. Further studies are needed to clarify apoptosis and cell biomarkers in SSCs during freezing and thawing.

In Vitro Culture of Ovine Spermatogonial Stem Cells: Effects of Grape Seed Extracts and Vitamin C

“…The culture environment is a static system in vitro, and the cells cultured require numerous nutrients and show a higher concentration of reactive oxygen species (ROS) (2). The ROS is involved in apoptosis signaling processes in organelles of the cells, such as mitochondria, under the regulation of Bcl-2 family proteins (3). Lipid peroxidation is caused by free radicals and results in DNA breaks and oxidization of proteins and important molecules.…”

“…It is also involved in many biological activities, including enzymatic reactions catalyzed by dioxygenases that use Fe 2+ and 2-oxoglutarate as co-substrates (13). Improving the culture media with sufficient vitamin C amounts leads to a more effective generation of pluripotent stem cells, somatic cells, SSCs (14), and somatic cell nuclear transfer animal embryos (3). Following the addition of vitamin C to the culture medium at certain concentrations, it can act as a growth promoter and increase DNA synthesis and cell proliferation.…”