# A 3D Printed Vitrification Device for Storage in Cryopreservation Vials
###### Abstract
Most sperm vitrification studies have been constrained to the adoption of tools that were initially designed for applications other than sperm, such as the Cryolopo (Hampton Research, Aliso Viejo, CA, USA) originally designed for crystallography [7; 8], the Cryotop(r) (KITAZATO, Valencia, Spain) designed for the vitrification of human oocytes and embryos [9; 10], pipette tips for liquid transfer [11], cryopreservation straws (IMV Technologies, L'Aigle, France) for the equilibrium freezing of sperm [12], and inoculation loops for microbiology [11]. Although these tools can be used for vitrification purposes, they can be expensive (e.g., the Cryotop(r) costs more than $20/device for 2-\(\upmu\)L samples) and are difficult to redesign, customize, securely label, and efficiently store. Three-dimensional (3D) printing combined with open-source sharing mechanisms is a promising strategy to address challenges in the development of sperm vitrification hardware. A version of this strategy has been successfully applied in software development and now is fueling a new movement in scientific hardware development [13]. Open hardware sharing allows maker community members to gain access to hardware designs through online platforms for free. Thanks to the increasing accessibility of 3D printers, user community members can download, fabricate, and assemble designs and devices for a low cost instead of purchasing expensive proprietary equipment or repurposing existing tools. In addition, members of user communities and maker communities can contribute to design changes for modifications and improvements, facilitating community-level standardization. In recent years, this potential has been explored for the application of 3D printing in cryobiology applications [14]. A 3D printed cryopreservation device was developed to freeze small batches (several dozens) of samples in French straws (0.25 mL and 0.5 mL) or cryovials (50-100 \(\upmu\)L) with standardizable cooling rates controlled by a modular assembly with liquid nitrogen vapor in Styrofoam boxes [15] and shipping dewars [16]. A 3D printed motorized controlled cooling conveyor device (CCCD) was developed for the continuous cryopreservation of non-batched sperm samples [17]. The feasibility of 3D printing for sperm vitrification application was demonstrated with simple 3D printed vitrification configurations [18; 19] featuring a single-piece loop that could load the sperm samples in the form of thin films. Based on this prototyping, an upgraded 3D printed vitrification device was developed with practical functions, including vitrification, volume control, labeling, protection, and storage within the existing straw storage systems [20]. All of these designs addressed the main problems of film formation and stability, rapid heat transfer to accommodate vitrification, permanent labeling, and protection and storage of the delicate devices and frozen films. In addition to French straws, another widely applied type of cryopreservation containers are vials with liquid-tight lids [21; 22]. A variety of commercially available vials have been developed to enable the high-throughput and efficient management of cryopreserved samples. To accommodate storage in vials, wider loops can be designed beyond the dimensions of the aforementioned studies (that were constrained by the width of 0.5-mL straws). The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials(VDCV) that can be integrated with existing cryo-vial storage systems. The specific objectives were to: (1) design and fabricate the VDCV, (2) evaluate the cooling rates by computer simulation, (3) evaluate the sample loading methods prior to freezing, (4) evaluate the vitrification occurrence, and (5) determine the volume capacity. The initial progress in the present study provides a foundation and guidance for further community-driven efforts in the development of vitrification devices with efficient storage systems.