Three-Dimensional Printing of Vitrification Loop Prototypes for Aquatic Species

Abstract

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.

On‐Site Capabilities of a Mobile Laboratory for Aquatic Germplasm Cryopreservation

“…; Tiersch et al. ). These devices could easily be incorporated into training programs, facilitating community development and allowing smaller user groups to develop cryopreservation programs.…”

“…As new user group communities are formed, the application of standardized protocols for sample preparation, cryopreservation, and quality control could be uniformly adopted for a wide range of purposes. Recently, three-dimensional printing has provided a means of fabricating low-cost cryopreservation devices (Tiersch and Monroe 2016;Hu et al 2017;Tiersch et al 2018). These devices could easily be incorporated into training programs, facilitating community development and allowing smaller user groups to develop cryopreservation programs.…”

Addressing Reproducibility in Cryopreservation, and Considerations Necessary for Commercialization and Community Development in Support of Genetic Resources of Aquatic Species

“…After suitable designs become adopted they can be coalesced into standardized, cheap and widely available versions. New technologies such as 3-dimensional printing can be used in all three of these developmental phases (Tiersch and Monroe 2016; Tiersch et al 2018) to provide inexpensive, customizable, and widely available devices by electronic sharing of design files. Stock centers would be effective central locations to develop, test, and promulgate such devices to improve reproducibility, coordinate overall cryopreservation programs including training and web-based resources, and to strengthen community identity and activities (more on this is presented below).…”

“…It is, therefore, worth consideration of online communities as a potential tool that can benefit aquatic species cryopreservation in terms of community development and reproducibility. A 3-D printing community is especially relevant to cryopreservation due to the ability to rapidly prototype and test cryogenic devices made especially for aquatic species (e.g., Hu et al 2017; Tiersch et al 2018).…”

“…This latter device (a bacteriological inoculation loop), which has been used to vitrify sperm and produce live young in a live-bearing fish species (Cuevas-Uribe et al 2011) clearly offers more promise for applied use and would warrant further study to improve its performance. Even better would be research addressing the customized production and testing of 3-D printed devices for vitrification at a material cost of around US$0.01 each (Tiersch et al 2018).…”