# A 3D Printed Vitrification Device for Storage in Cryopreservation Vials
## 2 Materials and Methods  
### Design and Fabrication of Prototypes with Various Dimensions  
The overall design goal was to create a device that could suspend a thin film of sperm sample within a loop that could fit into Nunc(tm) Bank-It(tm) cryogenic storage vials (Thermo Fisher Scientific, Waltham, MA, USA) for sample storage and management. These vials are routinely used in cooling and storage of sperm at the Zebrafish International Resource Center (ZIRC) and other research laboratories (personal communication with ZIRC). Each unit of vial (Figure 1A) featured a tube with QR codes at the bottom and a cap with an inner hollow thread for fastening inside the tube, an O-ring for sealing, and an outer hollow grip at the bottom for handling and mounting of the decapping tools. Two components were designed for the VDCV: a vitrification component (Figure 1B) with a loop to form thin films and a handling component (Figure 1C) with a cylindrical shaft to facilitate sample processing (Figure 1D,E). The design considerations are detailed below. The vitrification component should fit in 0.5-mL Bank-It(tm) vial tubes with an internal diameter of 6.8 mm and length of 20 mm and could be fastened inside the hollow thread (with an inner diameter of 4.8 mm). The handle should be fastened to the hollow grip (with an inner diameter of 6.7 mm). Based on the preliminary trials of sample loading and vitrification feasibility, the loops of the vitrification component were designed as three lengths (8, 10, and 12 mm) of the major axis (referred to as 'loop lengths') and four lengths (2, 2.5, 3, and 3.5 mm) of the minor axis (referred to as 'loop widths'). Previously reported vitrification devices had a loop width of 2.1 mm to accommodate storage within straw sleeves [20]. Because the higher loop widths were designed in the present study to take advantage of a larger cross-sectional area of vials, support mechanisms were evaluated to stabilize films prior to freezing. Within the loop, three variations of film support configurations were designed: open loop, loop with axial support, and loop with transverse support. A total of 36 configurations (3 loop lengths \(\times\) 4 loop widths \(\times\) 3 supports) were developed for operational testing.  
To support the open-fabrication availability of designs, consumer-level software and 3D printers were used. Designs of 3D modeling were created using a free online platform for Computer-Assisted Design (CAD) (Onshape, PTC Inc., Boston, MA, USA). The 3D renderings of the prototypes were converted to stereolithography (STL) files and imported into free slicing software (Ultimaker Cura V4.6, Ultimaker, Utrecht, The Netherlands) to define the printer settings (Supplemental Table S1) in G-Code format. The settings were loaded onto a fused deposition modeling (FDM) 3D printer (SOVOL, Sv01, SOVOL 3D(r), NOVA SILK ROAD SARL, Paris, France) printed with a PLA filament (ZYLTech Engineering, Houston, TX, USA). For a batch of 5 duplicates (Figure S1) of each vitrification component, printing took 8-15 min and \(<\)US$0.01 (\(\sim\)0.4 g of PLA filament). The handling component took 1.5-2 h and \(<\)US$0.5 (\(\sim\)10 g) to print. As such, about US$0.5 material cost was required to print a the handling component of the VDCV and an additional US$0.5 to print 250 pieces of the vitrification components (loops).