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Abstract: Background: In cryopreservation, cooling rate is a dominant factor that influences the survival of cells. Box-in-box (BIB) was recently developed as a reliable, cooling rate controlled and cost-effective cooling device. However, the intrinsic heat transfer characteristic still needs to be further specified for the best of design and application of the device. Method: The freezing process of samples inside BIB is simulated by developing a one dimensional heat transfer model in which fixed-grid technique is used to solve the solidification problem of the ternary cryopreservation media (water, NaCl and cryoprotectant). Based on the model, several critical factors, including supercooling temperature, structural parameters and application conditions, are evaluated respectively. Several cell free experiments were also conducted to validate the model. Results: It was demonstrated that BIB method can achieve uniform and consistent cooling of samples, and the theoretical and experimental results fit quite well. Further analysis reveals that several structural parameters (such as the dimension of insulation layer) and application conditions (such as the cryoprotectant concentration and the sample volume) have significant effect on the freezing process of sample. Thus the design and application of BIB should be carefully conducted to achieve the desired cooling rate. Conclusion: The theoretical model is reasonable for the BIB system. It provides an effective tool to determine the detailed structural parameters when designing BIBs, and it can also be used as a good support for the application of BIB systems. [Copyright &y& Elsevier]