Table 1.
Comparison of the high-glycerol (HGM) and low-glycerol (LGM) methods for the cryopreservation of human RBCs.
Fig 1.
Optimization of an adapted glycerolization procedure and comparison to the standard reported literature method.
(A) Diagram of the standard glycerolization protocol and our adapted method, designed to minimize sample processing time and allow collection/processing/storage of smaller RBC aliquots. In the standard glycerolization method, RBC volumes were added dropwise resulting in a final solution that was 40% w/v glycerol; in the adapted method, RBCs were added as a bolus, resulting in a final 40% v/v glycerol solution. All centrifugation was performed at 1,500g, 5min, at 4°C. (B) Whisker-plot comparing RBC lysis resulting from the addition of buffer/glycerol (60% v/v glycerol buffer stock added to samples) at different temperatures to pRBCs in a one step (bolus addition; 300μl pRBC to 600μl buffer/glycerol stock) or two step (300μl pRBCs to 300μl buffer to 300μl buffer/glycerol stock = step one, followed by addition of further 900μl buffer/glycerol stock = step two) process. Note, both procedures resulted in a 40% v/v glycerol final. Additionally in this experiment, no samples were frozen. Glycerol solutions were added and then samples immediately centrifuged and lysis measured. (C) Whisker-plot comparing the order of addition of pRBC, buffer, and buffer/glycerol (60% v/v glycerol buffer stock added to samples) in the two step glycerolization procedure, on the amount of lysis following sample freezing at -80°C and thawing for 2 minutes at 37°C. Order of addition of components is highly important. (D) Whisker-plot comparing RBC lysis following sample freezing and thawing, for the standard (40% w/v glycerol final) and our adapted (40% v/v glycerol final) glycerolization procedure. Mean RBC lysis was not different between the two methods; however, our adapted glycerolization procedure was 40% more time efficient (n = 9 matched samples from 3 individual blood donors).
Fig 2.
Comparison of the standard and our adapted deglycerolization procedure.
(A) Diagram of the standard deglycerolization protocol and our adapted method, designed to reduce RBC lysis. Samples (40% v/v glycerol final) were thawed (2min, 37°C), then centrifuged (1,500g, 5min, 4°C). For equilibration, samples were left undisturbed for 3min at RT. Circled numbers identify sampling points for the graphs presented in B-D. (B) Sample osmolality was measured at various stages of the standard and adapted deglycerolization methods. The adapted method slowed osmolality reduction during de-gycerolization, thereby protecting cells from lysis (n = 3–8 samples from 3 individual blood donors). (C) Total cumulative RBC lysis measured at different stages of the standard and adapted deglycerolization methods. RBC lysis was significantly reduced using the adapted deglycerolization method (n = samples, shown on graph. Minimum number of individual blood donors = 4). (D) Intracellular RBC glycerol measured at different stages of the standard and adapted deglycerolization methods. Both methods result in total glycerol removal (> 99.9%) (n = 4–6 samples from 3 individual blood donors).
Table 2.
Characteristics of the buffers used in sample glycerolization and deglycerolization.
Fig 3.
RBC morphological imaging (scanning electron microscope; SEM) and comparison of osmotic fragility, deformability, and ex vivo adhesion in matched fresh and deglycerolized RBCs.
(A) Illustration of the characteristic biconcave disc morphology of fresh RBCs. Following glycerol addition, the majority of RBCs become swollen and spherocytic. After thawing, RBCs remain encapsulated in a glycerol matrix. Following deglycerolization RBCs return to their original morphology. (B) NaCl induced osmotic lysis in matched fresh, deglycerolized, and deglycerolized RBCs exposed to a prolonged buffer incubation (n = 5 or 10 individual donors, shown on graph). NaCl induced osmotic lysis increased immediately following deglycerolization, but returned to the original fresh phenotype following prolonged incubation (1 hour) of RBCs in the final deglycerolization buffer. (C) Half maximal effective concentration (EC50) increased immediately following deglycerolization, in comparison to fresh RBC samples (p < 0.05). However, prolonged incubation (1 hour) of RBCs in the final deglycerolization buffer at 37°C restored the original fresh RBC phenotype (n = 5 or 10 individual donors, shown on graph). (D) Shear induced maximal RBC deformability (Elongation Index maximum; EImax) was not different between matched fresh and deglycerolized RBCs (exposed to the prolonged buffer immersion) (n = ±6 individual donors). (E) The shear stress (Pa) at which RBC demonstrated 50% elongation (SS50) was also not different between matched fresh and deglycerolized RBCs (exposed to the prolonged buffer immersion) (n = 6 individual donors). (F) RBC adhesivity to endothelium was determined ex vivo. A trend was observed toward a decrease in RBC adhesivity in the deglycerolized RBCs, across all sheer stresses > 1 dyne/cm2, that did not reach statistical significance.
Fig 4.
Comparison of O2 dissociation curves (ODCs) and RBC hypoxic vasodilatory response (HVD) from matched fresh and deglycerolized RBCs, and NO content of matched fresh RBCs, frozen RBCs (without glycerol), cryopreserved RBCs (without glycerol removal), and cryopreserved RBCs (deglycerolized), measured by photolysis:chemiluminescence.
(A) Raw ODC curves from matched fresh and deglycerolized RBCs in BIS TRIS buffer (BIS TRIS 50mM, NaCl, 100mM), pH to 7.2, 7.4, and 7.6. Inset is the whole ODC curve (n = 10 individual donors). No difference was observed between fresh and deglycerolized RBCs. (B) Hill coefficient, a measure of cooperativity, was not different between matched fresh and deglycerolized RBC samples across the 3 different pH values. (C) Bohr plot demonstrates no change between matched fresh and deglycerolized RBC samples. (D) Representative single traces (from rings demonstrating similar PE constriction responses) of the relaxation responses from fresh vs deglycerolized RBCs under hypoxia (~ 1% O2)—Arrow indicates injection point for RBCs. (E) Relaxation responses of fresh and deglycerolized RBCs (as a percentage of maximal constriction) are not different in a hypoxic organ chamber bioassay of endothelial intact rabbit aortic rings. (F) Total RBC NO content was not significantly different between any of the conditions, although freezing appeared to slightly augment NO levels. (G) Iron nitrosyl hemoglobin (HbFeNO) content remained unchanged in all conditions. (H) S-nitrosohemoglobin (SNO-Hb) significantly increased upon freezing, independent of glycerol presence (p < 0.05). Data reported as NO:Hb (tetramer basis).
Fig 5.
Comparison of metabolomic steady state phenotypes and metabolic fluxes in response to oxidant stress in fresh and matched deglycerolized RBCs.
(A) Tracing experiments were performed by incubating RBCs with [1,2,3-13C3]glucose, a heavy substrate that can be metabolized in RBCs through (B) the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (generating isotopologue M+3) or through the pentose phosphate pathway (PPP), generating isotopologue M+2 of lactate, (C) owing to the release of the first carbon atom of heavy glucose in the form of 13CO2 at the oxidative phase reactions of the PPP. (F) Schematic overview of glucose uptake and consumption in RBC supernatants and cells, in fresh, or deglycerolized RBCs, either untreated, or treated with the superoxide generator (SOTS-1), (D) demonstrating in both RBCs and media similar glucose consumption rates. (E) Total lactate, i.e., sum of lactate isotopologues (unlabeled M+0, M+3 generated via glycolysis, or M+2 generated via the PPP), in fresh and DG RBCs, either untreated or treated with the superoxide generator (SOTS-1), demonstrating in both RBCs and media similar lactate accumulation. (G and H) Schematic overview of expected isotopologues and relative ratios of lactate, from glycolysis (EMP, M+3 isotopologue) or PPP (M+2) and relative ratios in fresh and DG RBCs, either untreated, or treated with the supoxide generator SOTS-1. (H) Fresh RBCs were characterized by significantly higher levels of supernatant–but not intracellular—M+2 lactate. Conversely, deglycerolized RBCs were characterized by faster glycolysis than fresh RBCs. Lower lactate M+2 export in deglycerolized RBCs following SOTS-1 was observed, while intracellular glycolysis to pentose phosphate pathway ratios were comparable between fresh and deglycerolized RBCs following treatment with SOTS-1. Continuous lines indicate median, while dashed lighter lines are representative of interquartile ranges, according to the color legend left. Symbols indicate p<0.05 + = fresh vs DG; & = fresh + SOTS vs DG + SOTS; * = fresh vs fresh + SOTS; # = DG vs DG + SOTS.
Fig 6.
Unsupervised analysis of metabolomics data via PLS-DA, with dendrograms for time course hierarchical clustering and heat maps, visually indicating a time course and SOTS dependency of the metabolic phenotypes observed in fresh RBC and matched DG RBCs.
(A) The impact of time and SOTS treatment informed sample clustering along principal components 1 and 2 (explaining 21.9 and 17.4% of the total variance, respectively). Both (A) PLS-DA and (B) hierarchical clustering analyses confirmed that control and DG groups fell into the same clusters at the tested time points, both in the absence and presence of SOTS. (C) Heat maps visually indicate the time course and SOTS dependency of the metabolic phenotypes observed both in fresh RBC and DG groups.
Fig 7.
Dorsal window chamber set up for the assessment of in vivo hemoglobin O2 saturation, RBC velocity and vascular adherence of transfused fresh or matched deglycerolized RBCs.
(A) Image of the dorsal window chamber set up. (B and C) No difference was observed in mapped hemoglobin O2 saturation, or (D) RBC velocity in the dorsal skinfold window chamber model, between the transfusion of fresh or deglycerolized RBCs. (E) A modest, but statistically insignificant difference was observed on in vivo adhesivity of transfused RBCs between fresh and deglycerolized RBCs from the same donors.