Effect of Warming Process on the Survival of Cryopreserved Human Peripheral Blood Mononuclear Cells.

It is well known that the warming process is a critical step in cell cryopreservation, affecting the survival rate of the cryopreserved cells. However, there is a lack of understanding and optimization of the warming process for the cryopreserved human peripheral blood mononuclear cells (PBMCs) that are greatly needed for the cellular/immune therapies worldwide. In this study, the effect of the warming process on cryosurvival of the PBMCs was investigated, resulting in a recommendation of an optimal warming method. In the experiments, all PBMC samples were cooled by a fixed slow cooling process and stored in a liquid nitrogen tank. The frozen samples were then warmed in water baths with stirring at various temperatures, 37°C, 42°C, and 65°C, respectively. After thawing, PBMC's viability as well as phenotypic and functional analyses were performed and evaluated. It was shown that a relatively rapid warming process at 65°C in a water bath with stirring generated a significant improvement of cell viability, recovery, and functionality of the cryopreserved PBMCs. In addition, interferon-γ and interleukin-2 secretion were much higher in PBMCs thawed at 65°C than that in 42°C and 37°C, respectively. This study suggests that a rapid warming process at 65°C in a water bath should be used to replace the current conventional warming approach at 37°C.

Improvement of a Simple and Cost-Effective Passive Cooling Rate-Controlled Device for Cell/Tissue Cryopreservation.

The currently used commercial cooling-rate control device is the liquid nitrogen controlled rate freezer (LNF), which has some shortcomings such as high cost, high liquid nitrogen consumption, and potential operational risks in quality control. Based on thermophysical properties of new materials, we improved, manufactured, and optimized a reliable yet simple device named the "passive cooling rate-controlled device (PCD)" with real-time temperature tracing. In this study, using the improved PCD we cryopreserved human umbilical vein endothelial cells (HUVECs) and compared the results with a standard commercial CryoMed LNF. The temperature profiles and cooling rates of the HUVEC samples in a cryopreservation solution (with dimethyl sulfoxide [DMSO] in 10% v/v concentration) were measured and automatically recorded by the PCD during the controlled cooling process. This study and experimental results showed that the HUVEC survival rates after cryopreservation using the PCD have no significant difference from those using the CryoMed LNF and that the improved PCD is a user-friendly, reliable, and low-cost device to ensure an optimal slow cooling rate ranging from -0.5 to -1°C/min for the cryopreservation. Considering the advantages of low cost, durability, reliability, and no liquid nitrogen consumption for the cooling process, it is concluded that the PCD is an excellent controlled cooling device to achieve a desired optimal cooling rate for cell/tissue cryopreservation.