A 3-D printed vitrification device integrated with French straws.

The goal of this work was to develop prototypes of open-hardware vitrification devices for sperm cryopreservation that can be integrated with existing straw platforms. The open-hardware Vitrification Device for French Straws (VD-FS) is low-cost, customizable, 3-D printable, standardized, and allows long-term sample storage and identification. The feasibility was shown for vitrifying and storing samples with multiple configurations. The results can be improved by design alternation and evaluation of various vitrification solutions. This is the first complete open-hardware vitrification device that can be integrated with existing French-straw storage systems, providing a foundation for future community-level modifications and improvements.

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.

Standardized Assessment of Thin-film Vitrification for Aquatic Species.

Ultra-rapid cooling under the appropriate conditions will produce vitrification, a glass-like state used to cryopreserve small sample volumes, but there are a number of major technical drawbacks impeding application of vitrification to germplasm of aquatic species. These include a lack of suitable devices, and poor reproducibility and comparability among studies due to a lack of standardization. We used 3-dimensional (3-D) printing to produce a viewing pedestal coupled with a classification system to rapidly assess frozen film quality of vitrification loops. Classification time declined with practice from 2.1 ± 0.3 sec to 1.5 ± 0.2 sec (after 200 assessments), and assessments were consistently made in < 2.5 sec. Classifications should be reported with representative images allowing harmonization for quality control. This approach permits rapid classification and can be applied for development of methods including evaluation of vitrification solution components, concentrations of solution and target cells, and configurations and volumes of new devices. Future studies should address the custom fabrication of 3-D printed vitrification devices for use with aquatic species and other applications.