Improving cryopreservation outcomes of small biological samples through ultra-fast convective cooling and warming

Project Summary/Abstract Cryopreservation of oocytes, embryos and other small biological samples plays a key role in medicine, in biomedical research, and in assisted reproduction of humans, domestic animals, biomedical research animals, and endangered species. Hundreds of thousands of genetically distinct strains of species including pigs, fruit flies, and zebrafish are maintained for biomedical research purposes. Many species/strains are maintained as live stocks or are propagated by cloning because reliable and cost-effective oocyte/embryo cryopreservation technology is not available. Such technology would provide benefits including simplification of protocols, protection against genetic drift from generation to generation, preservation of culled stocks, and protection against disasters. Current cryopreservation approaches involve soaking samples in cryoprotectant solutions, cooling by hand plunging in liquid nitrogen, and warming by hand plunging in a warm solution. Poor post-thaw survival and development outcomes are primarily associated with ice formation, although osmotic shock during soaks, cryoprotectant toxicity, sample microfracturing, and phase transitions and chemical changes in lipids can contribute. Some improvement in cryopreservation outcomes has been achieved using alternative warming methods based on, e.g., electromagnetic energy absorption by injected nanoparticles. This project aims to develop and validate cryopreservation technology that automates soaking, cooling, and warming; that optimizes conventional convective heat transfer to increase cooling rates in liquid nitrogen and warming rates in aqueous solutions by factors of 102 over current practice; that can eliminate ice formation and associated damage and allow cryoprotectant concentrations to be reduced; that reduces cooling and warming times toward ~1 millisecond, allowing other damage mechanisms to be outrun; and that improves reproducibility of key steps, facilitating optimization of other factors important to successful outcomes. This work is informed by studies of ice formation in aqueous solutions during fast cooling and warming; by MiTeGen’s expertise and technology base in ultra-fast cooling using liquid nitrogen; by expertise in diagnostics including time-resolved x- ray diffraction and high speed optical imaging that can detect and quantify ice formed in oocytes/embryos during cooling and warming; and by extensive preliminary studies on bovine oocytes and embryos demonstrating the feasibility of fully ice-free cryopreservation and how ultra-fast cooling improves developmental outcomes. Technology to be developed includes a flexible robot-based workstation with stations for soaking, liquid removal, ultra-fast cooling, and ultra-fast warming; and ultra-low thermal mass sample supports that maximize convective cooling and warming rates while retaining samples through all steps. This technology will be validated, first, through measurements of cooling/warming rates and ice formation in aqueous cryoprotectant solutions and, second, through studies of ice formation in and post-thaw survival, development, and transcriptomes of bovine and porcine embryos.