Variation in the osmotic characteristics of aging red blood cells: insights for cryopreservation optimization

Cryopreservation is a long-term storage strategy for maintaining the quality of red blood cells (RBCs) used for clinical and industrial purposes. However, cryopreservation can induce osmotic stress, leading to a 15 to 20% loss of RBCs during freezing/thawing and cryoprotectant addition/removal. This study investigated how the biological aging of RBCs influences their osmotic characteristics. We hypothesized that biologically older RBCs (O-RBCs) would exhibit diminished osmotic features compared to biologically younger RBCs (Y-RBCs), thereby contributing to their loss during cryopreservation. Seven red cell concentrates (RCCs) were pooled, and their density profile was determined using Percoll separation. Y-RBCs and O-RBCs, representing the extremes of the density spectrum, were isolated. Rheological parameters (O_hyper, EI_max, EI_hyper, K_EI) were measured with laser ektacytometry. Osmotic fragility and hemolysis tests were performed to assess subpopulation responses to osmotic stress. Water (L_p) and solute permeability (P_s) of these subpopulations, in addition to unseparated RBCs (U-RBCs), were measured using stopped-flow spectroscopy. Aliquots of Y- and O-RBCs were differentially labeled with biotin and spiked back into a pooled unit. The pooled unit was split into five, and cryopreserved via a high glycerol/slow-cooling method, a standard method adopted by international blood banks. Glycerolization and deglycerolization were conducted using the Haemonetics ACP 215 Automated Cell Processor. Flow cytometry was used to assess the recovery of biotinylated RBCs (BioRBCs) before glycerolization and following post-thaw deglycerolization on days 1 and 14. O-RBCs exhibited the highest rigidity (K_EI) and lowest O_hyper, EI_max, and EI_hyper. Osmotic hemolysis and osmotic fragility indicated that O-RBCs exhibited superior tolerance to hypotonic solutions than Y- and U-RBCs (P < 0.05). O-RBCs demonstrated significantly higher L_p values than Y-RBCs across hypo- and hypertonic conditions (P < 0.05). O-RBCs had the highest P_s during deglycerolization (P < 0.05). Y- and O-BioRBCs showed a stable trend throughout the 14 days of hypothermic storage (1-4°C) post-deglycerolization, with no significant difference between subpopulations. The study revealed that biological aging is associated with alterations in the osmotic and rheological properties of RBCs. Despite notable differences in osmotic characteristics, under the test conditions, survival rates of Y- and O-BioRBCs remained comparable after 14 days of hypothermic storage. These findings potentially support that tailoring cryopreservation protocols to specific RBC subpopulations can improve recovery rates and make cryopreservation more clinically efficient and broadly applicable.