3-D printed customizable vitrification devices for preservation of genetic resources of aquatic species

Abstract

Sperm vitrification as an alternative approach to conventional cryopreservation (equilibrium freezing) allows quick and low-cost sample preservation and is suitable for small-bodied aquatic species with miniscule testis, fieldwork at remote locations, and small-scale freezing for research purposes. The goal of this present study was to develop operational prototypes of 3-dimensional (3-D) printed vitrification devices with innovative components that can provide comprehensive functionalities for practical repository development for aquatic species. The design featured an elongated loop to suspend a thin film of sperm sample in cryoprotectant, a retractable sleeve to protect the vitrified samples and allow permanent labeling, a handle to facilitate processing and storage, and a shaft with annular grooves to guide positioning of the protective retractable sleeve. To span a wide range of sample capacities and configurations, a total of 39 different configurations (3 loop lengths ×13 loop heights) were fabricated by 3-D printing with the thermoplastics polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS). A total of 86 devices were fabricated with ABS filament with a print failure rate of 9%, and 97 devices were fabricated with PLA filament with a failure rate of 20 %. Major types of printing failures included disconnected loops, insufficient build surface adhesion, stringing, and inconsistent extrusion. The sample volume capacity ranged from 1 to 47 μL and had linear relationships to the loop lengths and layer numbers. Vitrified samples were observed in 10-mm and 15-mm loops fabricated with PLA and ABS but not in 20-mm loops. This study demonstrated the feasibility of development of standardized low-cost ($0.05 material cost) devices fabricated by 3-D printing with practical functions including vitrification, volume control, labeling, protection, and storage within conventional systems. These prototypes can be further developed, standardized, and used to assist development of germplasm repositories to protect the genetic resources of aquatic species by user groups such as breeders, hatcheries, aquariums, and researchers.

Introduction

Development of germplasm repositories to protect the genetic resources of aquatic species has been hindered by several factors over the past 70 years including an almost complete focus on cryopreservation research and protocol development. Other problems include a lack of approaches for standardization, and the requirement to adapt equipment and supplies developed for livestock and human medicine for use with fish and shellfish. New fabrication technologies such as 3-dimensional (3-D) printing can provide expanded access to CAD-CAM capabilities, and open new opportunities for custom design and production of standardizable devices directly based on the needs of aquatic user communities. Inexpensive devices such as these can be distributed as open-source files to facilitate application, and to support and focus protocol development, ensuring that high-quality material can be made available to centralized germplasm repositories.

Because of the current lack of repository development, the utility of cryopreservation remains largely unrealized for aquatic species in multiple areas including genetic improvement for aquaculture (Blackburn, 2011; Hu et al., 2011), stock enhancement for wild fisheries (Riley et al., 2004; Tiersch, 2004), protection of genetic diversity in imperiled species (Liu et al., 2018; Wayman et al., 2008), and storage and distribution of tens of thousands of research lines of biomedical research models (Torres et al., 2017; Yang and Tiersch, 2009). Cryopreserved sperm has been incorporated into germplasm repositories for protection and management of genetic resources in other species such as livestock (Purdy et al., 2016), but that is because they have moved past protocol research into application, often by utilization of engineering approaches.

Efforts in application of engineering technologies for sperm cryopreservation have primarily focused on conventional cryopreservation (‘equilibrium freezing’) methods. A critical factor determining the success of equilibrium freezing is to identify and achieve ideal cooling rates (e.g., 5−40 °C/min) during freezing. Control of cooling rate requires specialized equipment, which can cost tens of thousands of dollars for computer-programmed types or several thousand dollars for other types.

An alternative and relatively new method for sperm cryopreservation is vitrification, by which liquid is cooled at > 1000 °C/min (‘rapid cooling’) to transform into an amorphous solid (glass) phase without the formation of crystalline ice (Cuevas-Uribe et al., 2017; Rall and Fahy, 1985). The rapid cooling can be obtained simply by plunging a thin film (e.g., several μl loaded on loops) or droplets (e.g., on plates or strips) of sample into liquid nitrogen. As such, vitrification allows low-cost sample preservation (Magnotti et al., 2018) and is suitable for: (1) small-bodied species with miniscule sample volumes, (2) fieldwork at remote locations where equipment or electricity are not accessible, and (3) small-scale freezing for research purposes. For example, swordtails and guppies (family Poeciliidae) are popular ornamental and aquaculture species in the U.S. and typically provide < 5 μL of sperm from each male (Huang et al., 2009; Yang, 2009), and thus vitrification could be an ideal method for preserving sperm of these species for genetic management purposes (Cuevas-Uribe et al., 2011b).

There are several major limitations of existing devices (i.e., with specialized vitrification functions) and tools (i.e., designed for applications other than vitrification) used in sperm vitrification. Firstly, most commercial vitrification devices previously reported were designed for freezing of mammalian oocytes and embryos, and thus only accommodate small sample volumes (e.g. < 2 μL) for sperm loading. For example, the Cryotop® devices (KITAZATO, Valencia, Spain), designed for vitrification of human oocytes and embryos, were used in sperm vitrification of Eurasian perch (Perca fluviatilis) and European eel (Anguilla anguilla) (Kása et al., 2017). However, only 2 μL of sperm suspension could be loaded onto each device (Marco-Jiménez et al., 2016). Secondly, devices specifically designed for sperm vitrification are often medical devices intended for human clinical application, and thus these devices are costly. For example, the Cryotop® costs more than $20/device. The Sperm VD device (Berkovitz et al., 2018) designed for sperm vitrification with storing and labeling mechanisms costs $60 and can only load about 1 μL of sample per device. Thirdly, non-specialized tools have been adopted for sperm vitrification. For example, a study of sperm vitrification of channel catfish (Ictalurus punctatus) evaluated various options (Cuevas-Uribe et al., 2011a), such as pipette tips (originally for liquid transfer), sperm cryopreservation straws (for equilibrium freezing of semen) cut at various angles, and inoculation loops (for microbiology). Although these tools can help reduce costs and some of them can provide limited functionality for operation and sample recovery, they lack the capability to be customized, standardized, securely labeled, and efficiently stored.

Recently, the increasing availability of consumer-level 3-D printing makes it possible to rapidly prototype and fabricate devices at a low cost. This technology has been introduced to the field of cryobiology (Hu et al., 2017; Tiersch and Monroe, 2016) and repository development for aquatic species (Tiersch and Tiersch, 2017). Previous work has demonstrated the feasibility of using 3-D printed loops with a material cost of $0.01/unit to perform sample vitrification (Tiersch et al., 2019). Given the feasibility of vitrification within 3-D printed loops (a single component) the next step is design and test operational devices (multiple integrated components), with additional features to achieve practical capabilities and functionalities, such as handling, sorting, labeling, and storage. The goal of the present study was to develop and test operational prototypes of low-cost 3-D printed sperm vitrification devices with innovative elements that can provide comprehensive functionalities for practical repository development for aquatic species. The specific objectives were to: (1) design component prototypes and operational prototypes; (2) evaluate fabrication feasibility with consumer-grade 3-D printers; (3) evaluate the relationship of sample volume capacity with various configurations, and (4) evaluate the feasibility of operational prototypes to achieve vitrification. The innovation of these operational prototypes can provide a foundation for further performance testing, and divergent modifications, and ultimately standardization (as a long-term goal) based on the needs of user communities.