# A 3D Printed Vitrification Device for Storage in Cryopreservation Vials
## 2 Materials and Methods
### Comparison of Designs with Computer Simulation  
To assist in CVCD support configuration design and facilitate prototyping, computational fluid dynamics (CFD) software (Autodesk CFD 2019, Autodesk, San Rafael, CA, USA) was used to compare the cooling rates and temperature distributions of designs with different loop support configurations. The 3D models of the vitrification loops were created in Autodesk Inventor (Autodesk), converted to STL files, and imported to Autodesk CFD. The following material properties were assumed to not vary with the temperature and were assigned for polylactic acid (PLA) based on the manufacturer's specifications, including 0.13-W/m thermal conductivity, 1.29-g/cm\({}^{3}\) density, 1800-J/kg\({}^{-\text{k}}\) specific heat, and 0.92 emissivity. The initial temperature of the loops was set at 20 \({}^{\circ}\)C to simulate room temperature prior to plunging into liquid nitrogen. A boundary condition of 200 \({}^{\circ}\)C (liquid nitrogen temperature) steady-state temperature was assigned to all surfaces to simulate the temperature during exposure (e.g., rapid plunging) to liquid nitrogen. Automatic mesh sizing was initially uniformly applied and refined in the size adjustment from the default 1.0 to 0.2. The meshing element count for each design was above 1 million elements. To facilitate the computations, more complicated physical phenomena, such as phase transition, surface tension, and convection, were not considered in this study. Heat transfer models were applied, and the flow dynamics were disabled. The simulation process was run in a transient mode, with time steps of 0.001 s and an inner interval of \({}^{\prime}1\). The cooling rate was calculated as the time (min) to cool water to from 20 \({}^{\circ}\)C to \(-\)137 \({}^{\circ}\)C (136 K, the commonly acknowledged glass transition temperature of water) [23]. The physical properties of water were assigned to the films within loop structures. To simplify the simulation, the thickness of the films was set at 0.4 mm (flush with the upper and bottom surfaces of the loops) without taking into consideration the surface curvature caused by meniscus effects, liquid surface tension, and gravity. The cooling rates at 5 positions in the open loop design were sampled in the simulations, including the center position for open  
Figure 1: Vitrification Device for Cryo-Vials (VDCV) fabricated by 3D printing. (**A**) A 0.5-mL NuncTM Bank-ItTM cryogenic storage vial comprises a cap and a tube. (**B**) Vitrification components with three different configurations (from left to right): open loop, axial support, and transverse support. A handling component (**C**) with structures (**D**) to fit 0.5-mL Bank-ItTM cryogenic storage vials (**E**). loops (PO), centers of the two compartments divided by axial supports (PA1 and PA2), and centers of the two compartments divided by transverse supports (PT1 and PT2). Cooling rates were averaged for PA1 and PA2 as PA and PT1 and PT2 as PT. Evaluation of the Effects of Sample Handling Methods and Device Configurations on Film Retention Rates  
Different sample loading methods and loop configurations can affect the retention of sample films on the device prototypes prior to freezing. Deionized (DI) water (without cryoprotectant) was used as a model fluid in this experiment. Loops of the VDCV were submerged vertically into DI water for >2 s. Upon removal from the liquid, the loops were held horizontally or vertically (as two different handling methods) for 5 s to observe whether the water films were retained or not at the 5 th second. Film loading and observation were repeated 10 times, and the film retention rate was calculated as: (times of retention/10) \(\times\) 100%. Three loop lengths and four loop widths were tested with 5 replicates (printed in a single batch) for each configuration.