Theoretical optimization of the removal of cryoprotective agents using a dilution-filtration system.

BACKGROUND: In the cryopreservation of blood, removing cryoprotectants from the cryopreserved blood safely and effectively is always being focused on. In our previous work, a dilution-filtration system was proposed to achieve the efficient clearance of cryoprotectants from the cryopreserved blood. METHOD: In this study, a theoretical method is presented to optimize the diluent flow rate in the system to further reduce the osmotic damage to red blood cells (RBCs) and shorten the washing time necessary to remove cryoprotective agents (CPAs), based on a discrete mass transfer concept. In the method, the diluent flow rate is automatically adjusted by a program code in each cycle to maximize the clearance of CPAs, whereas the volume of RBCs is always maintained below the upper volume tolerance limit. RESULTS: The results show that the optimized diluent flow rate can significantly decrease the washing time of CPAs. The washing time under the optimized diluent flow rate can be reduced by over 50%, compared to the one under the fixed diluent flow rate. In addition, the advantage of our method becomes more significant when the blood flow rate is lower, the dilution region volume is larger, the initial CPA concentration is higher, or the cell-swelling limit set by the system is smaller. CONCLUSION: The proposed method for the dilution-filtration system is an ideal solution for not only guaranteeing the volume safety of RBCs but also shortening the washing time of CPAs. In practice, the optimization strategies provided here will be useful in the rapid preparation of cryopreserved blood for clinical use.

Statistical estimation of red blood cell osmotic damage during cryoprotective agent removal from cryopreserved blood.

Statistical estimation of the osmotic damage of red blood cells (RBCs) during the removal of cryoprotective agents (CPAs) from cryopreserved blood has been a very difficult issue. In this paper, the discrete mass transfer model developed in our previous work is modified to study the volume variation of individual RBCs and thereby to estimate the osmotic damage of all RBCs statistically during CPA removal by the dilution-concentration method we proposed recently. The model is validated with respect to the experimental results either with or without RBCs. Then, it is used to investigate the effects of blood volume, hematocrit, blood and diluent flow rates on the osmotic damage of RBCs, as well as the washing time of CPAs. Our results show that both the increase of blood flow rates and the decrease of diluent flow rates can bring about a reduction in osmotic damage of RBCs; however, only the former can cause a decrease in the washing time of CPAs. The blood volume could also affect the osmotic damage of RBCs. For a given flow condition, there could exist an optimal blood volume range for the dilution-concentration system. The effect of blood volume could be alleviated by an increase in the dilution region volume. In addition, the osmotic damage of RBCs decreases as the hematocrit decreases. Therefore, in practice, the increase of blood flow rates is the best solution to reduce both the osmotic damage of RBCs and the washing time of CPAs simultaneously. A lower hematocrit in the cryopreserved blood and/or longer tubing in the dilution region are also recommended to achieve better performance for the dilution-concentration method.