Cryopreservation has become an important and accepted tool for long-term germplasm conservation of animals and plants. To protect genetic resources, repositories have been developed with national and international cooperation. For a repository to be effective, the genetic material submitted must be of good quality and comparable to other submissions. However, due to a variety of reasons, including constraints in knowledge and available resources, cryopreservation methods for aquatic species vary widely across user groups which reduces reproducibility and weakens quality control. Herein we describe a standardizable freezing device produced using 3-dimensional (3-D) printing and introduce the concept of network sharing to achieve aggregate high-throughput cryopreservation for aquatic species. The objectives were to: 1) adapt widely available polystyrene foam products that would be inexpensive, portable, and provide adequate work space; 2) develop a design suitable for 3-D printing that could provide multiple configurations, be inexpensive, and easy to use, and 3) evaluate various configurations to attain freezing rates suitable for various common cryopreservation containers. Through this approach, identical components can be accessed globally, and we demonstrated that 3-D printers can be used to fabricate parts for standardizable freezing devices yielding relevant and reproducible cooling rates across users. With standardized devices for freezing, methods and samples can harmonize into an aggregated high-throughput pathway not currently available for aquatic species repository development.

Vitrification is a method of cryopreservation that freezes samples rapidly, while forming an amorphous solid (''glass''), typically in small (lL) volumes. The goal of this project was to create, by three-dimensional (3D) printing, open vitrification devices based on an elliptical loop that could be efficiently used and stored. Vitrification efforts can benefit from the application of 3D printing, and to begin integration of this technology, we addressed four main variables: thermoplastic filament type, loop length, loop height, and method of loading. Our objectives were to: (1) design vitrification loops with varied dimensions; (2) print prototype loops for testing; (3) evaluate loading methods for the devices; and (4) classify vitrification responses to multiple device configurations. The various configurations were designed digitally using 3D CAD (Computer Aided Design) software, and prototype devices were produced with MakerBot Ò 3D printers. The thermoplastic filaments used to produce devices were acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA). Vitrification devices were characterized by the film volumes formed with different methods of loading (pipetting or submersion). Frozen films were classified to determine vitrification quality: zero (opaque, or abundant crystalline ice formation); one (translucent, or partial vitrification), or two (transparent, or substantial vitrification, glass). A published vitrification solution was used to conduct experiments. Loading by pipetting formed frozen films more reliably than by submersion, but submersion yielded fewer filling problems and was more rapid. The loop designs that yielded the highest levels of vitrification enabled rapid transfer of heat, and most often were characterized as being longer and consisting of fewer layers (height). 3D printing can assist standardization of vitrification methods and research, yet can also provide the ability to quickly design and fabricate custom devices when needed.

Although aquatic species cryopreservation protocols have been studied around the world over the past 60 yr., germplasm repository development efforts and commercialization have begun only recently. The goal of this project was to develop a self-contained mobile laboratory for on-site high-throughput cryopreservation of aquatic species. The objectives of this study were to: (1) identify how a mobile laboratory would function in different operational scenarios, (2) customize an enclosed cargo trailer to function as a mobile laboratory, (3) evaluate the laboratory layout and ability of cryopreservation equipment to operate from generator power, and (4) document the investment costs for private and public groups to integrate a mobile laboratory into an existing cryopreservation facility at three levels of automation and estimate the total cost per trip based on hypothetical assumptions for two scenarios (aquaculture production and repository development). There were three operational designs identified for the mobile laboratory: (1) self-contained work inside the unit using generator power, (2) work inside the unit using external facility power, and (3) using the equipment inside of a host facility. on level of automation. Overall, a commercial-scale mobile laboratory was developed that can assist current germplasm activities and support future repository and industry development, and the layout information provided can help others to design and build comparable units.

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