# A 3D Printed Vitrification Device for Storage in Cryopreservation Vials
## 2 Materials and Methods
### Comparison of Designs with Computer Simulation
Evaluation of the Effects of Plunging Orientation and Device Configurations on Vitrification Occurrence  
Based on the film retention experiment, different orientations during the sample plunging into liquid nitrogen were evaluated for their effect on the vitrification. A 3D printed sample plunging device was developed (Figure S2) that could hold loops at horizontal or vertical orientations to the liquid nitrogen surface and standardize the distance of descent and ascent with a sliding track. To evaluate the quality of vitrification formation, a standardized visual classification system was adapted as described below [18; 20]. After submerging in liquid nitrogen for about 2 s, the samples were removed and transferred to an observation station (Figure S2). The observation station was positioned in a liquid nitrogen vapor environment to extend the observation time prior to sample thawing. Frozen films in loops were precisely positioned in front of 2D barcodes and visually examined. A previous standard approach [18; 19] classified the vitrification outcomes into six categories by the visibility of the barcodes: 'No sample', 'Film failure', 'Fractured', Zero (opaque and crystalline)', 'One (translucent and partially vitrified)', and 'Two (transparent and vitrified)' (only 'Zero', 'One', and 'Two' were included in the statistical analyses). To facilitate the statistical analysis and training process, these six categories were grouped as: (1) \"No vitrification' (including 'No sample' and 'Film failure' samples); (2) 'possible vitrification' (including 'Fractured', 'Opaque', and 'Translucent' films), or (3) 'Full vitrification' (including transparent films). A maximum time for assessment was set at \(\leq\)3 s to ensure that the classifications were assigned before the films began to thaw. Vitrification was also evaluated with an existing vitrification solution used for aquatic species (20% Hanks' balanced salt solution, 40% methanol, 20% methyl glycol, and 20% 1,2 propanediol) [20]. All of the loops filled with this viscous solution retained their films prior to plunging into liquid nitrogen. To evaluate the effects of device configurations on vitrification, the three film support designs (open loop, axial support, and transverse support) were tested with loops with 10-mm lengths, and the support design with the best performance was selected to compare the effects of the three different loop lengths (8, 10, and 12 mm). The two plunging orientations (vertical and horizontal) were evaluated with the four different loop widths (2.0, 2.5, 3.0, and 3.5 mm) and three film support designs. Five different loops for each configuration were used as replicates with 10 testing runs for each loop. The occurrence rate of each vitrification classification was calculated as: (Occurrence times/10) \(\times\) 100%. The effect of different loop heights (only 0.4 mm was evaluated in this study) was not investigated, because reports have shown that 0.2-0.4-mm loop heights had the highest efficiencies in producing high-quality vitrification, but the printing quality of 0.2-mm loops was not consistent [20].