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The cooling process is critical for the cryopreservation of human hematopoietic stem cells (HSCs). Currently, programmed freezing methods and uncontrolled cooling methods are in use, both having obvious disadvantages. In this article, a novel device termed Box-in-Box (BIB) was developed and evaluated by in vitrocryopreservation tests in 2 different operation modes (“against-side” mode for Group I (n 10), and “in-middle” mode for Group II (n 10), respectively), and compared with an uncontrolled cooling method (Group III (n 7), Styrofoam boxes) as well as a conventional programmed freezer method (Group IV (n 10), CryoMed TM 1010, Cryogenic Tech., FL). Recorded temperature profiles of samples cryopreserved with BIB show that a consistent cooling procedure with a rate around −1°C to −3.5°C/min can be achieved during their transfer from room temperature to an −80°C freezer. Statistical analysis of the stem cell population recovery, survival, and colony generation recovery shows that there is no significant difference (P> 0.26) among the methods using the BIB or programmed freezer (Group I, Group II, and Group IV), and their related deviations are smaller than the uncontrolled cooling rate method (Group III). Methods using the BIB (Group I and Group II) generated significantly better cell survival rate (P< 0.01) than the uncontrolled cooling rate method (Group III). The results indicate that the controlled cooling rate methods (BIB or CryoMed PF) are more consistent and reliable for clinical use. Considering the advantages of low cost, durability, and no liquid nitrogen consumption for the cooling process, the BIB can be a good alternative to the programmed freezers for the cryopreservation of HSCs.