Studies On Process Of Human Red Blood Cells Loaded With Trehalose Before Lyophilization And Characteristics Of Rehydrated-Lyophilized Human Red Blood Cells

Compared to traditionary method of blood preservation, lyophilized red blood cells (RBCs) have many merits: stable quality on room temperature, easy to rehydrate, enormously reduced weight, easy to transport and sterilize, so lyophlilization may be the most effective preserving method of RBCs. To maintain the integrity of membrane is the key and nodus in lyophilization of RBCs. Based on judicious selection of cryoprotective agents (CPAs), we must exert the effect of CPAs. As the preferred CPA trehalose has distinct effect which can well protect organism or bioactive molecule in dried, frozen,hot and humid conditions, so it is widely used to preserve cells. Because of non-osmosis to membrane trehalose can not permeate the membrane of RBCs generally. But more and more researches demonstrated when entering RBCs trehalose could show effective protection to membrane and intra-cellular protein. Based on this idea the anthor carried out three studies:1. Optimization of trehalose loading in red blood cells before Freeze-drying. This study showed Regularity of trehalose loading in red blood cells and evaluate damage of RBCs during trehalose loading process. The sulfuric-anthrone reaction was used to determine the intracellular trehalose concentration loaded by RBCs, meanwhile we evaluate extracellular free hemoglobin and ATP level of loaded RBCs. The results showed that with increase of incubation temperature, duration and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. The study proved trehalose can enter RBCs by osmosis nonequilibrium and phase change of membrane liquid. The result also showed that 4℃ stored period of RBCs can not effect the loading efficiency of trehalose but can increase the hyperosmosis damage of RBCs.Based on the loading of trehalose and the changes of free hemoglobin, MCV and ATP level, it is concluded that the optimal condition of loading trehalose is choicing fresh RBCs which were incubated in 800mmol/L trehalose solution at 37℃ for 8 hours, resulting in a effective cytoplasmic trehalose concentration. Considering that absorption effiency of trehalose keep company with RBCs' damage, so to better the method of loading to improve the loading efficiency and decrease haemolysis of RBCs is the next study idea.2. Effect of dimethylsulfoxide (DMSO) on human red blood cells protection during the period of loading trehalose before freeze-drying. To study the effect of different concentration of DMSO during the period of loading trehalose, so we can optimize the compouding of loading solution. The study demonstrated that DMSO can carry trehalose so as to enter RBCs more easily which can increase the loading of trehalose. Based on the loading of trehalose and the changes of extracellular free hemoglobin, we determined the final adding concentration of DMSO is 6%. It is further showed that 6% DMSO can effectively increase cytoplasmic trehalose concentration, improve red cell deformability (RCD) and inhibit consumption of ATP during incubation, which can enhance the protection effect of trehalose on RBCs.3. Lyophilization of human RBCs loaded trehalose. The study showed after handled with trehalose-loading buffer adding 6% DMSO, rehydrated-lyophilized human RBCs can increase hemoglobin recovery of RBCs, improve RCD, enhance the stability of rehydrated RBCs when stored in 4℃ refrigerate and long-term preservation of lyophilized RBCs.