Design, alpha testing, and beta testing of a 3-D printed open-hardware portable cryopreservation device for aquatic species.

Efforts in development of germplasm repositories to preserve genetic resources of aquatic species are impeded globally by a lack of standardized, inexpensive, reproducible, and portable cryopreservation technologies. The present work demonstrates a 3-D printed standardizable freezing device that can be used with nitrogen vapor shipping dewars for on-site sperm cryopreservation for aquatic species and be distributed as open-source The SDPCD could hold 22 French straws (0.25-mL or 0.5-mL) and a quick-release ring design could eject straws directly into a canister inside a dewar by pressing a button after freezing. The final prototypes produced cooling rates of 1 to 64 °C/min for 0.25-mL straws, and 3 to 37 °C/min for 0.5-mL straws with material cost of US$3.5 for a single device and US$1,820-2,562 for batch production of 20 replicates. Progressing through design, prototyping, and testing was delineated to help guide development of other open-source devices within cryopreservation user communities.

On-Site Capabilities of a Mobile Laboratory for Aquatic Germplasm Cryopreservation.

Cryopreservation of genetic material can become an important tool for user groups in imperiled fishes, wild fisheries, aquaculture, and biomedical research. Persistent challenges within aquatic species cryopreservation are standardization and reliable collection of diverse, high quality samples. The overall goal of this study was to work with different user groups and cryopreserve sperm on-site at their facilities to evaluate the uses and challenges of a mobile laboratory with high-throughput and quality control capabilities comparable to those of a specialized centralized facility. The objectives were to demonstrate collection and cryopreservation of sperm of: 1) large-bodied freshwater Blue Catfish (Ictalurus furcatus) for aquaculture; 2) small-bodied freshwater Xiphophorus for biomedical and imperiled repository development, and 3) saltwater Red Snapper (Lutjanus campechanus) for wild fisheries research. Over the course of this project, the mobile laboratory traveled more than 4,000 km collecting germplasm from more than 650 male fishes. A total of 136 Blue Catfish were processed in 2015 and 2016 resulting in a total of 6,146 0.5-mL French straws. A total of 521 males from 11 different species in the genus Xiphophorus were processed over 4 d in 2015 resulting in a total of 488 0.25-mL French straws. And, a total of 17 Red Snapper males were processed during 2015 resulting in a total of 316 0.5-mL French straws. This is the first development of a mobile laboratory with high-throughput capability for aquatic species. User groups would no longer be limited to germplasm resources that can only be shipped as samples or transported as live animals to a central cryopreservation facility. Mobile laboratories create opportunities to collect higher quality germplasm, provide access to new species, and enable direct cooperation, including training, with a wide variety of user groups and applications.

Three-Dimensional Printing of Vitrification Loop Prototypes for Aquatic Species.

Vitrification is a method of cryopreservation that freezes samples rapidly, while forming an amorphous solid ("glass"), typically in small (µL) 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® 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.

Design and Cost Analysis of a Self-contained Mobile Laboratory for Commercial-scale Aquatic Species Cryopreservation.

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. The investment costs for a base-level mobile laboratory ranged between US$5670 and US$5787 for private groups and between US$5208 and US$5315 for public groups. With the addition of a range of automated processing equipment, total investment costs ranged from US$13,616 to US$103,529 for private groups and US$12,494 to US$94,891 for public groups. The total cost per trip to cryopreserve sperm of 59 blue catfish, Ictalurus furcatus, males to produce 6300 0.5-mL French straws was estimated to range from US$6089 to US$14,633 for private and between US$5703 and US$16,938 for public groups depending on the level of automation. Total cost per trip to cryopreserve sperm of 500 males of five different species in the genus Xiphophorus to produce 641 0.25-mL French straws was estimated to range from US$6653 to US$7640 for private and US$7582 to US$8088 for public groups depending 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.