The Protection Of Small Molecurlar Sugars On Lyophilized Human Red Blood Cells

Lyophilization of human red blood cells has great implication on clinical transfusion and therapy of war wound. At present, the main methods to preserve human red blood cells include 4℃storage and cryopreservation at -80℃. The protective effect of these methods is excellent, but there are some significant shortages in these methods. The storage time at 4℃is short and easy contamination by microbes can occur. Although cryopreservation at -80℃can greatly prolong the storage time to about 10 years, this method needs a heavy ultra-low-temperature refrigerator or other storage equipments. In addition, after thawing, glycerol needs removal through complicated washing process. Compared with the conventional methods, lyophilization has the following advantages: room temperature storage, less weight, easy transportation.However, lyophilization may be difficult. Serious leakage of hemoglobin in red blood cells can occur during lyophilization and rehydration. Lyophilization seriously injures the membrane. Trehalose can increase the ability of membrane to tolerate drying. In this study, small molecular sugars (trehalose and glucose) and polymer were used as main lyoprotectants to lyophilize human red blood cells. At first, the sugar loading regularity and the effect of sugar loading process on structure and physico-chemical property of red blood cells were studied. Small molecular sugars were loaded into red blood cells through a combination of osmotic imbalance and phospholipids phase transition. Then glucose, trehalose, human serum albumin, and PVP were used as main protectants to evaluate the feasibility of lyophilization of red blood cells. The lyophilization conditions including the protectant concentration, vitrification degree, freezing temperature, shelf temperature, and the rehydration conditions were also optimized. The data showed: 1 the uptake of sugars in red blood cells has certain regularity. With increase of extracellular sugar concentration, elevation of incubatin temperatures, and prolonging of incubation time, the sugar loading efficiency was increased steadily. But the glucose loading efficiency was significantly more than the trehalose loading efficiency. At the same incubation condition, the injuries of trehalose on hemolysis and deformability of red blood cells were significantly more than that of glucose, but the ability of trehalose to maintain the asymmetrical distribution of membrane PS and increase osmotolerance of red blood cells was significantly higher than that of glucose. In addition, trehalose can decrease the peroxidative damage of membrane phospholipids during sugar loading process. Addition of trehalose in the glucose buffer can significantly increase osmotolerance of red blood cells, maintain the asymmetrical distribution of PS and decrease peroxidative damage. Moreover, glucose can mitigate hemolysis during trehalose loading process. The caspase-3 and caspase-8 have not been activated during apoptosis of human red blood cells induced by high glucose. In addition, leupeptin can efficiently inhibit PS exposure of red blood cells induced by high glucose. 2 during the optimization of the lyoprotectants, loading both trehalose and glucose into cytoplasma can significantly decreases the percent hemolysis and loss of metabolic function of red blood cells after lyophilization and rehydration. The DSC data showed with increase of the PVP concentrations, the onset temperature of crystal was decreased, which showed the vitrification degree of protective solutions was increased. With the increase of the vitrification degree, the percent hemolysis after rehydration was firstly decreased and then increased, which showed excessive vitrification might not be suitable for lyophilization. Moreover, human serum albumin also can provide protection for lyophilized red blood cells. 3 the ultra-low freezing temperatures can lead to increase of hemolysis of lyophilized red blood cells owing to second warming during prefreezing. When the shelf temperatures were lower than -30°C, the percent hemolysis of cells after lyophilization and rehydration was approximately 20% and there were not significant difference among the treating groups. 4 adding low concentrations of polymers in the rehydration solutions can increase survival of lyophilized red blood cells. In addition, higher temperature also can significantly decrease hemolysis after rehydration. 5 Approximately 80% lyophilized cells observed by TEM have intact membrane, but some cells still had an echinocytic shape and the hemoglobin of some cells had partially leaked away.In conclusion, this study showed it is feasible to lyophilize human red blood cells using small molecular sugars and polymers as main lyoprotectants. Although trehalose has supervier advantages on dried cells, the self characteristics of red blood cells lead to serious injuries during trehalose loading process. Sugar loading process causes serious cell injuries which in turn manifest themselves during subsequent lyophilization and rehydration. But combination of glucose and trehalose can increase intracellular sugar concentration and osmotolerance, inhibit PS exposure, and decrease peroxidative injuries. The future work will focus on finding a safe and efficient sugar loading process and deeply study the effect of lyophilization on ultrastructure of red blood cells.