With advances in biotechnology, the field of cryopreservation has been continuously developed and improved. Typical cryo-container was designed with minimal flow to avoid possible structural defects in LN2 tank, which has a higher thermal conductivity than vapor nitrogen tank. If cells are placed in typical cryo-container and stored in VN2 tank, cross-contamination can be prevented, but the cell viability after thawing may be reduced. The structure of typical cryo-containers is not optimized for vaporized nitrogen to flow quickly into the container and its circulation well. Therefore, we proposed new cryo-container models that can maintain mechanical strength while optimizing the fluid flow structure, and performed thermal–structural coupled field analysis on cryo-containers. We confirmed the cryo-containers by comparing the equivalent stress distributions formed around through holes and evaluating thermal equilibrium in the cryogenic steady-state through flow analysis. Prototype cryo-containers and typical cryo-containers were placed in VN2 tank for a period of time to observe temperature changes. As a result, the time it takes to reach the temperature equilibrium has been reduced to 55% level compared with the typical cryo-containers. Additionally, C2C12 and hADMSC cells were checked after storage under two temperature conditions (−80 and −196 °C). In both cell, viability, adhesion, and relative cell proliferation were improved by up to 15–20% in new containers compared to typical products. The developed container is expected to maintain stability well by being applied to storage and transportation of advanced medicines that require cryopreservation.