Cryopreservation of zebrafish larvae and embryos for biomedical research

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PROJECT SUMMARY Zebrafish are one of the most broadly used research model systems because they are easy to breed, manipulate, and image. It is this immense utility that has led to the creation of tens of thousands of transgenic, reporter, and mutant lines that have been instrumental in our understanding of biology, characterizing disease, and discovering new therapeutics, to name only a few. Despite zebrafish being relatively cheap to house, maintaining large stocks of live fish indefinitely is associated with facility space, personnel, and maintenance costs that also risks the loss of irreplaceable research lines due to catastrophic events and genetic modifications. In this capacity, a safe and reliable method for cryopreservation of zebrafish lines would provide a means to bank precious resources for the research community. Consequently, this project aims to develop a zebrafish embryo and larvae cryopreservation protocol that is compatible with different storage temperatures and to train external labs with an easy to implement protocol through extensive dissemination efforts. While the field has been relatively successful in establishing sperm banks for a range of fish, egg/embryo cryopreservation has been met with severe challenges, despite the clear advantages. Cryopreservation of sperm requires at least 6 months to generate an adult strain, in contrast to cryopreservation of embryos that would enable more rapid and direct use upon thawing. However, the two main cryopreservation approaches – slow cooling and vitrification – each have severe limitations that have remained insurmountable. Slow cooling uses low concentrations of cryoprotectant agents (CPAs) yet must compete with extensive ice formation that can damage cells. Alternatively, ice-free methods, such as vitrification, require high concentration of toxic CPAs and high cooling rates that limit scalability. Instead, we propose a relatively unexplored method termed interrupted cooling. Interrupted cooling uses low concentrations of CPAs as samples are slowly cooled to high subzero temperatures (~-20°C) with limited ice formation, before a rapid cooling protocol to deep cryogenic temperatures that converts the remaining unfrozen fraction into a vitreous-like state. This flexible protocol enables both a short-term preservation step at high subzero temperatures and a long-term preservation solution for zebrafish banking that is compatible with dry ice for shipment and liquid nitrogen for reliable in-house storage. This will be achieved by applying first principles in cryopreservation to design a storage solution and interrupted cooling protocol that will protect complex systems without adverse functional consequences (Specific Aim 1). These efforts will be enhanced by applying lessons from wood frogs in nature to improve zebrafish survival after thawing (Specific Aim 2). Finally, in Specific Aim 3, we will perform extensive dissemination efforts with non-study staff and create publicly available training tools for the broader community.