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

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 3h in a 37°C water bath, red blood cells were frozen in liquid nitrogen for 24h 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 400mM, 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 400mM, 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.

[1]  Yoshitaka Miyamoto,et al.  Improvement of Hepatocyte Viability after Cryopreservation by Supplementation of Long-Chain Oligosaccharide in the Freezing Medium in Rats and Humans , 2006, Cell transplantation.

[2]  P. Gikas,et al.  Hydroxyethyl Starch: Extracellular Cryophylactic Agent for Erythrocytes , 1967, Science.

[3]  Mehmet Toner,et al.  Beneficial effect of microinjected trehalose on the cryosurvival of human oocytes. , 2002, Fertility and sterility.

[4]  F. Levine,et al.  Trehalose expression confers desiccation tolerance on human cells , 2000, Nature Biotechnology.

[5]  T. Standl,et al.  Transfusion of Autologous, Hydroxyethyl Starch-Cryopreserved Red Blood Cells , 1997, Anesthesia and analgesia.

[6]  A. E. Oliver,et al.  Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification. , 1997, Cryobiology.

[7]  Ying Han,et al.  Improved preservation of human red blood cells by lyophilization. , 2005, Cryobiology.

[8]  E. Bremer,et al.  Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments , 1998, Archives of Microbiology.

[9]  J. Crowe,et al.  Preservation of mammalian cells—learning nature's tricks , 2000, Nature Biotechnology.

[10]  Ying Han,et al.  Changes of phosphatidylserine distribution in human red blood cells during the process of loading sugars. , 2006, Cryobiology.

[11]  S. N. Timasheff,et al.  The thermodynamic mechanism of protein stabilization by trehalose. , 1997, Biophysical chemistry.

[12]  A. D. Brown,et al.  Microbial water stress. , 1976, Bacteriological reviews.

[13]  J. Acker,et al.  Trehalose loading into red blood cells is accompanied with hemoglobin oxidation and membrane lipid peroxidation. , 2008, Cryobiology.

[14]  R. Williams.  The surface activity of PVP and other polymers and their antihemolytic capacity. , 1983, Cryobiology.

[15]  Jiexi Wang,et al.  Inhibition of high glucose-induced erythrocyte phosphatidylserine exposure by leupeptin and disaccharides. , 2008, Cryobiology.

[16]  Mehmet Toner,et al.  Intracellular trehalose improves the survival of cryopreserved mammalian cells , 2000, Nature Biotechnology.

[17]  Anil Dhawan,et al.  Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing , 2005, Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society.

[18]  M. Marchand-Arvier,et al.  In vitro study of the protective effect of trehalose and dextran during freezing of human red blood cells in liquid nitrogen. , 1997, Cryobiology.

[19]  A. Strøm,et al.  Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose , 1987, Archives of Microbiology.

[20]  J. Crowe,et al.  Human platelets loaded with trehalose survive freeze-drying. , 2001, Cryobiology.

[21]  A. Rowe.  Cryopreservation of Red Cells by Freezing and Vitrification – Some Recollections and Predictions , 2002, Transfusion Medicine and Hemotherapy.

[22]  J H Crowe,et al.  Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying , 1995, Applied and environmental microbiology.

[23]  Z. Török,et al.  Loading red blood cells with trehalose: a step towards biostabilization. , 2004, Cryobiology.

[24]  A. Rowe,et al.  Liquid nitrogen preservation of red blood cells for transfusion; a low glycerol-rapid freeze procedure. , 1968, Cryobiology.

[25]  A. Rinfret.  FACTORS AFFECTING THE ERYTHROCYTE DURING RAPID FREEZING AND THAWING , 1960, Annals of the New York Academy of Sciences.

[26]  J. Acker,et al.  Biopreservation of red blood cells: past, present, and future. , 2005, Transfusion medicine reviews.

[27]  C. Anuradha,et al.  Inhibition of lipid peroxidation, protein glycation and elevation of membrane ion pump activity by taurine in RBC exposed to high glucose. , 2003, Clinica chimica acta; international journal of clinical chemistry.

[28]  A. Tunnacliffe,et al.  Intracellular trehalose improves osmotolerance but not desiccation tolerance in mammalian cells , 2000, FEBS letters.

[29]  Ying Han,et al.  Effects of pre-freeze incubation of human red blood cells with various sugars on postthaw recovery when using a dextran-rapid cooling protocol. , 2009, Cryobiology.

[30]  Ana D. Lopez,et al.  Trehalose: A Cryoprotectant That Enhances Recovery and Preserves Function of Human Pancreatic Islets After Long-Term Storage , 1997, Diabetes.