# A 3D Printed Vitrification Device for Storage in Cryopreservation Vials
###### Abstract  
Sperm cryopreservation by vitrification is a promising approach for small-bodied animals such as zebrafish (_Danio rerio_). However, most vitrification tools adopted in aquatic research were initially designed for applications other than sperm (such as human embryo freezing) and, thus, pose challenges for adoption to sperm vitrification. Three-dimensional (3D) printing combined with open hardware sharing is an emerging strategy to address challenges in the development of cryopreservation tools. The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials (VDCV) that can be integrated with the existing vial storage systems. The VDCV combined the vitrification and handling components to achieve functions of sample handling, vitrification, storage, and identification. The vitrification component featured a base, a stem, and a loop. A total of 36 configurations with various loop lengths (8, 10, and 12 mm); loop widths (2.0, 2.5, 3.0, and 3.5 mm); and support structures (open, transverse, and axial) of the VDCD prototypes were designed, fabricated, and tested. Device handling orientations (horizontal and vertical holding angles prior to and during freezing) were also investigated. Computer simulations estimated that the cooling rate of the samples ranged from 0.6-1.5 \(\times\) 10\({}^{5}\)\({}^{\circ}\)C/min in all the configurations. Prior to freezing, loops with axial supports produced a minimum of 92% film retention. The overall trends of full vitrification occurrence were observed: horizontal plunging > vertical plunging, and axial support > transverse support and open loop. A loop length of 8 mm had the highest overall vitrification occurrence (86-100%). No significant differences (\(p\) = 0.6584) were shown in a volume capacity (5.7-6.0 \(\mu\)L) among the three supporting configurations. A single unit of VDCV can provide loading efficiencies of about 6 \(\times\) 10\({}^{7}\) sperm/vial, pooling of samples from 3-6 males/vial, and fertilization for 1800 eggs/vial. The VDCV are low-cost (<$0.5 material cost per unit) and can be customized, standardized, securely labeled, and efficiently stored. The prototypes can be accessed by user communities through open-fabrication file sharing and fabricated with consumer-level 3D printers, thus facilitating community-level standardization.  
v 2021 25 August 2021 28 August  
new method for sperm cryopreservation is vitrification, by which the liquid is cooled at >1000 \({}^{\circ}\)C/min ('rapid cooling') to transform into an amorphous solid (glass) phase without the formation of crystalline ice [2; 3]. The rapid cooling can be obtained simply by plunging a thin film (e.g., several \(\upmu\)L loaded on loops) or droplets (e.g., on plates or strips) of the sample into liquid nitrogen. Vitrification has several benefits over equilibrium freezing, including low cost, portability, and superior post-thaw sample quality [4; 5]. It is suitable for small sample volumes (e.g., <100 \(\upmu\)L) or small-scale freezing for research purposes.  
The development of inexpensive and varied sperm vitrification tools has been slow and hampers the wide application and research of this technology. Commercial sperm vitrification products are scarce. The Sperm VD device [6] designed for sperm vitrification with storing and labeling mechanisms costs $74 each ([[https://us.ivfstore.com/products/sperm-vd?variant=12851114180685](https://us.ivfstore.com/products/sperm-vd?variant=12851114180685)]([https://us.ivfstore.com/products/sperm-vd?variant=12851114180685](https://us.ivfstore.com/products/sperm-vd?variant=12851114180685)), accessed on 27 August 2021) and can only accommodate about 1 \(\upmu\)L of sperm sample per device.