SBIR-STTR Award

In the past decade laboratories around the world have produced tens of thousands of mutant transgenic and wild type zebrafish lines for a wide range of genetic and biomedical research Maintaining all of these valuable genotypes is expensive risky and beyond the capacity of even the largest stock centers Cryopreservation of zebrafish sperm eggs and embryos is vital to the strategy of NIH s Division of Comparative Medicine which envisions increased multi institutional research using animal models To date zebrafish sperm and eggs have been successfully cryopreserved but zebrafish embryos have not The main challenges with zebrafish embryo cryopreservation are the large size of the embryo which limits the rate at which the embryo can be externally cooled and warmed and a multi compartmental embryo with different permeabilities preventing uniform diffusion of cryoprotectant agents Methods and apparatus that enabled the long term storage and transport of cryopreserved embryos would address a critical need for zebrafish researchers This project will develop and optimize a gold nanoparticle GNP based rapid warming technology that has successfully generated viable zebrafish grown from a cryopreserved embryos Due to the large size of the embryo traditional warming mechanisms are too slow and result in the formation of ice crystals that damage the embryos and prevent them from being viable The key innovation of this project addresses this limitation in the warming step Injected GNP act as a distributed network of ultra efficient heaters that generate warming rates of millions of C min when illuminated with an infrared laser To achieve this ultrafast and reproducible warming stable low toxicity GNPs with strong absorption at the laser wavelength will be fabricated and their photothermal properties measured A micro injection technique developed at the University of Minnesota will be optimized to circumvent the permeability barrier to safely introduce cryoprotectant agents CPAs and GNPs into the yolk and chorion compartments Embryos will be rapidly cooled to prevent damaging ice formation and laser warming techniques will be optimized to increase embryo survival after thawing The combined foundational research of Dr Bischof and his collaborators along with the expertise in GNP synthesis and manufacturing at nanoComposix will allow for rapid optimization and commercialization of this technology Project Narrative Zebrafish are an important model organism with genes and organs that share many similarities with humans allowing researchers to use zebrafish to study and better understand a cancer and other diseases and test the efficacy of pharmaceuticals that could one day be used to treat humans Researchers would benefit from a technology that allowed them to freeze and store zebrafish embryos which has not been possible until now enabling improved storage transport and sharing of zebrafish variants This collaborative effort between nanoComposix and Prof John Bischof at the University of Minnesota seeks to optimize a new method enabling rapid freezing and thawing of zebrafish embryos followed by expansion of the technology to provide a universal platform for preservation of other organisms

Project Summary/Abstract Laboratories around the world have produced tens of thousands of distinct zebrafish lines that serve as model organisms for genetic and biomedical research that applies to human genetics and diseases. Maintaining all these valuable genotypes is expensive and beyond the capacity of even the largest stock centers. One solution is the cryopreservation of zebrafish sperm, eggs and embryos, an approach that would enable the strategy of increased multi- institutional research using animal models envisioned by the NIH Division of Comparative Medicine. Zebrafish sperm and eggs have been successfully cryopreserved, but technical challenges had prevented the cryopreservation of zebrafish embryos until recent success demonstrated by nanoComposix and Prof. John Bischof at the University of Minnesota. The main challenge with zebrafish embryo cryopreservation is the large embryo size, which limits the rate at which the embryo can be externally cooled and warmed. The innovative approach used by the applicants utilizes an injected formulation of gold nanoparticles that act as ultra- efficient heaters to generate heat internally when illuminated with an infrared laser. This technology demonstrated, for the first time, the successful cooling, cryogenic stabilization and rewarming of zebrafish embryos. Further development and commercialization of this successful proof-of-concept will address a critical need for zebrafish researchers and will form the basis for other genetic preservation applications in aquaculture and human reproduction, improving reproduction for societal, environmental and biodiversity needs. This project will further optimize the zebrafish embryo laser warming reagents, protocols, and equipment developed during the Phase I contract. The equipment will be beta tested by zebrafish research centers and a commercial system will be manufactured. The system will consist of a reagent formulation composed of a cryoprotectant agent and a gold nanoparticle, optimized to provide high photothermal conversion efficiency and minimal toxicity, and will be manufactured under a ISO 13485-compliant quality system. Reagent microinjection into the embryos and freezing and laser rewarming protocols will be refined to further improve embryo survival rates and will form the basis of training materials for end users. Finally, first-generation equipment will be developed, along with testing of automated, high-throughput methods for embryo cooling. Further technology development will provide a universal high throughput platform for preserving germplasm of other vertebrate and non-vertebrate organisms.

Public Health Relevance Statement:
Project Narrative Zebrafish are an important model organism with genes and organs that share many similarities with humans. Researchers to use zebrafish to study and better understand cancer and other diseases and to test the efficacy of pharmaceuticals that could one day be used to treat humans. There is a critical need for a technology that allows zebrafish embryos to be frozen and stored, in order to preserve zebrafish strains with important genetic traits.

Project Terms:
3-Dimensional; Address; Adult; Animal Model; Aquaculture; assisted reproduction; base; Biodistribution; Biodiversity; biomaterial compatibility; Biomedical Research; Chorion; commercial application; commercialization; comparative; Contracts; cryogenics; Cryopreservation; Crystallization; Development; Dimensions; Diploidy; Disease; efficacy testing; egg; Embryo; embryo cryopreservation; embryo preservation; Equipment; Feasibility Studies; Fishes; Formulation; Freezing; Generations; Genes; Genetic; Genetic Diseases; Genetic Research; Genome; Genotype; Goals; Health; Hour; Human; Human Genetics; human model; Ice; improved; innovation; interest; International; Laboratories; Lasers; Legal patent; Letters; Malignant Neoplasms; Measures; Medicine; Methods; Microinjections; Minnesota; nanoGold; nanoparticle; off-patent; Organ; Organism; Other Genetics; Performance; Pharmacologic Substance; Phase; Physiologic pulse; Polyethylene Glycols; preservation; prevent; Printing; Process; Protocols documentation; Publishing; Reagent; Reproduction; reproductive; Research; Research Personnel; Rewarming; sperm cell; success; Surface; Survival Rate; System; Techniques; Technology; technology development; Testing; Time; Tissue Preservation; Toxic effect; Training; trait; United States National Institutes of Health; Universities; Work; Zebrafish; zebrafish genome