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

Abstract

BackgroundIn 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.MethodIn 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.ResultsThe 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.ConclusionThe 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.

Fatigue damage to pig erythrocytes during repeated swelling and shrinkage

“…Consequently, the volume of cells cannot be returned to that under isotonic conditions. These results are similar to the volume regulation obtained during the processes of CPA addition and removal [15,28]. The results in the present work imply that the leakage of intracellular substances in the process of unloading CPAs may occur even when the swelling volume of cells is maintained below the so-called safe upper volume tolerance limit.…”

“…However, because of its time-consuming, complex operation; lower cell recovery rate; and mechanical stress damage [21,22], scientists are exploring the washing routines of the variety of washing machines [15]. In addition, various alternative approaches have been also proposed, mainly including the dialysis-based method [23][24][25] dilution-filtration method [26][27][28][29], and theoretical work has been performed [30]. In the literature, most of the work focuses on the effects of the diluent addition time, the environmental temperature, the CPA type, the CPA concentration and the cell polydispersity on the volume excursion, the osmotic tolerance limits, and the cell membrane integrity [6,18,[31][32][33][34][35].…”

“…In the process of the removal of CPAs from cryopreserved-thawed cells samples, cells undergo repeated swelling and shrinkage due to the repeated change in the extracellular osmolality, especially when the dialysis-based method or the dilution-filtration method is used [23][24][25][26][27][28]36,37]. As a result, the repeated mode may cause cells to suffer from fatigue injury, such as membrane phospholipid reorganization [38], potassium leakage [10,14], and cytoskeletal damage [39].…”

Effect of the Polydispersity of RBCs on the Recovery Rate of RBCs during the Removal of CPAs

“…To optimize the methods mentioned above, many theoretical models have also been developed [ 18 – 25 ]. In these models, almost without exception, the three parameters of RBCs, such as the cell volume at isotonic condition, the cell surface area, and the osmotically inactive cell volume, were all assumed to be identical.…”

“…In the centrifugation method, a single cell was often studied to get optimal conditions for all cells. In the dialysis-based or dilution-filtration method, the blood was divided into discrete units so that the effect of the randomness of RBCs entering the washing system on the osmotic damage to RBCs and the washing time necessary to remove CPAs could be investigated; however, the cells were still considered the same [ 20 , 24 , 25 ]. In fact, the polydispersity of RBCs, that is, the difference between RBCs, exists widely [ 26 ].…”