Addition of oligosaccharide decreases the freezing lesions on human red blood cell membrane in the presence of dextran and glucose

Abstract

Although incubation with glucose before freezing can increase the recovery of human red blood cells frozen with polymer, this method can also result in membrane lesions. This study will evaluate whether addition of oligosaccharide (trehalose, sucrose, maltose, or raffinose) can improve the quality of red blood cell membrane after freezing in the presence of glucose and dextran. Following incubation with glucose or the combinations of glucose and oligosaccharides for 3 h in a 37 °C water bath, red blood cells were frozen in liquid nitrogen for 24 h using 40% dextran (W/V) as the extracellular protective solution. The postthaw quality was assessed by percent hemolysis, osmotic fragility, mean corpuscle volume (MCV), distribution of phosphatidylserine, the postthaw 4 °C stability, and the integrity of membrane. The results indicated the loading efficiency of glucose or oligosaccharide was dependent on their concentrations. Moreover, addition of trehalose or sucrose could efficiently decrease osmotic fragility of red blood cells caused by incubation with glucose before freezing. The percentage of damaged cell following incubation with glucose was 38.04 ± 21.68% and significantly more than that of the unfrozen cells (0.95 ± 0.28%, P < 0.01). However, with the increase of the concentrations of trehalose, the percentages of damaged cells were decreased steadily. When the concentration of trehalose was 400 mM, the percentage of damaged cells was 1.97 ± 0.73% and similar to that of the unfrozen cells ( P > 0.05). Moreover, similar to trehalose, raffinose can also efficiently prevent the osmotic injury caused by incubation with glucose. The microscopy results also indicated addition of trehalose could efficiently decrease the formation of ghosts caused by incubation with glucose. In addition, the gradient hemolysis study showed addition of oligosaccharide could significantly decrease the osmotic fragility of red blood cells caused by incubation with glucose. After freezing and thawing, when both glucose and trehalose, sucrose, or maltose were on the both sides of membrane, with increase of the concentrations of sugar, the percent hemolysis of frozen red blood cells was firstly decreased and then increased. When the total concentration of sugars was 400 mM, the percent hemolysis was significantly less than that of cells frozen in the presence of dextran and in the absence of glucose and various oligosaccharides ( P < 0.01). However, when both glucose and trehalose were only on the outer side of membrane, with increase of the concentrations of sugars, the percent hemolysis was increased steadily. Furthermore, addition of oligosaccharides can efficiently decrease the osmotic fragility and exposure of phosphatidylserine of red blood cells frozen with glucose and dextran. In addition, trehalose or raffinose can also efficiently mitigate the malignant effect of glucose on the postthaw 4 °C stability of red blood cells frozen in the presence of dextran. Finally, addition of trehalose can efficiently protect the integrity of red blood cell membrane following freezing with dextran and glucose. In conclusion, addition of oligosaccharide can efficiently reduce lesions of freezing on red blood cell membrane in the presence of glucose and dextran.