Thermally conductive graphene-based nanofluids, a novel class of cryosolutions for mouse blastocysts vitrification

Limited heating and cooling rates have long been recognized as bottlenecks in improving embryo cryopreservation. As a result, efforts to achieve higher heat transfer rates gave rise to milestones like open cryodevices and minimal media loading. A crucial but commonly ignored variable is heat conduction by cryosolutions. The low heat conductivity of the aqueous media surrounding embryos slows down cooling and heating rates of the embryo, imposing the risk of preventable damages. In this study, we introduce a novel thermally conductive cryosolution based on graphene oxide nanoparticles and test its performance against conventional sucrose-based solutions for vitrification of mouse blastocysts. Replacing sucrose with graphene oxide brought about similar re-expansion, hatching, and implantation rates of post-vitrification embryos while also preventing an array of cellular and molecular stresses. Our results showed significantly reduced oxidative stress, characterized by control-level expression of Sod1 and significant downregulation of Sod2 transcription when graphene oxide was used instead of sucrose. This molecular response was in agreement with the reduced level of reactive oxygen species produced in vitrified/warmed embryos using graphene-based solutions. The downstream impacts of this stress reduction manifested in significant downregulation of two major pro-apoptotic genes, Bax and Trp53, down to the same level as fresh embryos. Interestingly, embryos maintained their spherical shape during dehydration in graphene-based solutions and did not "collapse" when shrinking, like in sucrose-based solutions. These results provide new insights into the benefits of thermally conductive cryosolutions and showcase the potential of graphene oxide as a cryoprotectant in embryo vitrification.