Zebrafish (Danio rerio) is a powerful model in biomedical research and laboratories around the world have produced tens of thousands of mutant and transgenic lines. Maintaining these valuable genotypes as live fish is expensive, risky, and beyond the capacity of stock centers. As such, cryopreservation has become a necessity and most of these genetic resources are now maintained as samples of inconsistent quality frozen with rudimentary techniques. Quality control has not been practiced in any systematic way, reproducibility is poor, and protocols are not standardized. It is common to have problems and failures in fertilization resulting in lost lines that need to be recreated, causing facilities to waste considerable time and effort. This is largely due to the false notion that neglecting quality control saves time and money. However, rather than being reduced, these costs are shifted downstream through wasted storage space and reduced fertilization. This pervasive lack of quality control has placed the substantial investments in biomedical research at great risk. Therefore, our long-term goal is to provide inexpensive, universally available and systematic quality control leading to development of community-based standards for cryopreservation. This will enable reliable contributions from individual laboratories to large comprehensive repositories providing protection for genetic resources pivotal in biomedical research. To achieve this, we propose to improve reliability and efficiency by providing routine access to reproducibility and standardization through continued development of microfabricated (?laboratory on a chip?) and 3-dimensional (3-D) printed devices encompassing all process steps from sample collection through fertilization.
The Specific Aims are to: 1) Develop and test microfabricated devices that can be used by research laboratories at low effort and cost to improve assessment and study of sperm concentration and motility with respect to the effects of these factors on reproducibility and the overall success of cryopreservation. 2) Develop and test 3-D printed devices that can be used to improve the reproducibility of the freezing process. These devices will address conventional cryopreservation and vitrification, and be useful for single samples and pooled batches. 3) Perform biological testing of these devices to refine design and function and improve the reproducibility of quality assessment to enable research laboratories to back up lines or submit them to stock centers and germplasm repositories. This will provide a community-based approach for protection of genetic resources through systematic incorporation of devices, guidelines and standards applicable across a full range of activity scopes and scales.
Aquarium fish models are rapidly transforming genetic research of human disease, and as thousands of new research lines become available each year, we must develop the abilities and resources necessary to systematically preserve them in repositories for biomedical research and improvement of human health. The most significant problems constraining repository development for these fish are the lack of reliability and efficiency in cryopreservation capabilities caused by the absence of quality control, reproducibility, and standardization which prevent the coupling and integration of activities among research laboratories and stock centers, jeopardize maintenance of valuable lines, and produce costly inefficiency and duplication of efforts that slow research. Given that these perils multiply as the pool of cryopreserved samples is expanded through the continued use of current rudimentary methods, it is imperative to improve reliability and efficiency by providing routine access to reproducibility and standardization as we propose through continued development of microfabricated (?laboratory on a chip?) and 3-dimensional (3-D) printed devices.
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