3-D printing provides a novel approach for standardization and reproducibility of freezing devices

Abstract

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.

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

“…With enough user groups contributing, repositories could be supplied in aggregate with thousands of standardized samples, establishing much larger and more diverse repositories than are currently possible with the few available centralized cryopreservation facilities (Hu et al. ). Moreover, user community development can lead to the exchange of ideas and the development of new techniques and devices produced by emerging technologies, such as three‐dimensional printing, that could further increase standardization and harmonization throughout user groups and applications.…”

“…Recently, three‐dimensional printing has provided a means of fabricating low‐cost cryopreservation devices (Tiersch and Monroe ; Hu et al. ; Tiersch et al. ).…”

“…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.…”

Standardized Assessment of Thin‐film Vitrification for Aquatic Species

“…Future studies should address fabrication of 3-D printed vitrification devices, to specifically customize them for use with aquatic species and other applications. Three-dimensional printing with polylactic acid filament is especially suitable for prototyping and fabrication of devices intended for cryogenic applications (Tiersch and Monroe 2016) and offers tremendous opportunity to improve protocols and to assist development of community-based standards due to increasingly widespread ability to 3-D print design files shared over the internet (Hu et al 2017). …”

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

“…The manual scenario assumed that no automated high‐throughput equipment was utilized. French straws were hand filled and sealed by two operators, and 30 straws were frozen at a time in liquid nitrogen vapor using a polystyrene box and a three‐dimensional floating platform (Hu et al ). The semi‐automated scenario assumed that operators were hand filling and sealing straws, but a programmable freezer was utilized that could freeze as many as 2760 samples per freeze.…”