An Experimental Studies On Lyophilization And Rehydrated Characteristics Of Human Red Blood Cells

As freeze drying(lyophilization)preservation has many significant virtues than many other long-term preservation methods,it has become one new research hotpot of long-term preservation of cells.So lyophlilization may be the most effective preserving method of RBCs.The factors that influence the recovery rate of the freeze dried cells includes:the disposal of the RBCs before lyophlilization,the composition and concentration of the lyoprotective agents(LPAs),the methods of the addition and removal of the agents,the process of freezing and drying,and the rehydration.To maintain the integrity of membrane and the physiological function is the key and nodus in lyophilization of RBCs.All the work in this thesis will be explored around the topics of the trehaiose loading in RBCs before freeze-drying and its effects on the physiological and biochemistry properties of the RBCs,the selection of the LPAs,the influential factors in the process of lyophlilization,the rehydration conditions effects on the recovery rate of the RBCs.The freeze drying procedure and rehydration methods of human erythrocytes were optimized.1.Trehalose loading in RBCs before freeze-drying and its effects on the physiological and biochemistry properties of the RBCs.In the first part of the study showed an efficient method for loading RBCs with trehalose and evaluated the effects on the physiological and biochemistry properties of the RBCs,certificated that the RBCs could still carrying out there physilolgical functions after the trehalose loading.In this study the samples were divided into two groups:the control group(without loading RBCs in trehalose buffer),and the test group(loading RBCs in trehalose buffer).Anthrone reaction was used to determine the amount of the intracellular trehalose,meanwhile we evaluate extracellular free hemoglobin of loaded RBCs.Flow cytometry was used to assay the membrane integrality of the RBCs.The results showed trehalose loading in RBCs had closed correlation with the temperature,duration and extracellular trehalose concentration,that with increase of incubation temperature,duration and extracellular trehalose concentration,the loading of trehalose in RBCs also increased.After the trehaose loading,the counting,MHC,MCHC,the activity level of ATP,G-6-PD,SOD,2,3-DPG of the test group maintained close to the levels of the control group(p＞0.05).Flow cytometry data demonstrate that incubation of RBCs in a hypertonic trehalose solution results in a fraction of cells with different complexity that attached to little Annexin-V-FITC,and that it can be removed by washing and resuspending theRBCs in an iso-osmotic medium.Based on the loading of trehalose and the changes of physiological and biochemistry index,The method provides efficient uptake of the trehalose is choosing fresh RBCs which were incubated in 800 mmol/L trhalose at 37℃for a time span of 7h, resulting in a efficient intracellular trehalose concentrations of about 52mmol/L.The physiological and biochemistry stability and membrane integrality could be retained. This study provides an important first step in long-term preservation of RBCs.2.Optimization of LPAs and lyophilization procedures in human RBCs freeze-drying.In the second part,the parameters that affect the recovery of the rehydrated RBCs, including different concentrations of protective agents,the cooling rate before lyophilization,shelf's minimum temperature,temperature lifting rate of the main drying and secondary drying in lyophilization procedures,were studied.On the basis of above-mentioned parameters,we choose 0.8mol/L lycerol+15% PVP40+150mmol/L Terhalose+2%BSA(pH7.3)as the LPAs in post experiments. Liquid nitrogen ultrarapid cooling was used in lyophilization,i.e.to spurt the liquid nitrogen on the bottles to equilibrate them,then put the bottle on the shelf which has been precooling to -70℃.The optimized lyophilization procedure is:in the main drying, set the shelf temperature as-70℃1h.-60℃2h.-50℃2h.-40℃2h.-30℃2h.-25℃2h.-20℃2h.-15℃2h;in the secondary drying,set the shelf temperature as -10℃1h. 0℃1h.10℃2h.Set the drying chamber internal pressure as 150m Torr～200mTorr (about 20Pa～27 Pa).Set the condensator temperature as-85℃.The whole drying procedure lasting about 19h.3.Primary study of lyophilizated human RBCs' rehydration.The injury during rehydration is another very important reason that causes low recovery rate of lyophilized RBCs.So in the third part,the rehydration conditions that influence the recovery of the lyophilized RBCs,including different rehydration solution,the rehydration temperature,volume change rate of the RBCs in this stage(i.e. the lyophilized RBCs were rehydrated by the vapor firstly),were studied.Each changes of physiological and biochemistry properties of the RBCs were detected.(1)The influence of different rehydration conditions were:the solution of 10% (w/v)PVP40 in PBS has the best effect;RBCs recovery rate increased with increase of rehydration temperature,in 37℃,we get the best result;pre-rehydration in the vapor could raise the RBCs's recoveryrate,cause the MCV and RDW close to the fresh RBCs.(2)The deformability of the rehydrated RBCs decreased,but the activity level of ATP,G-6-PD,SOD,2,3-DPG of the rehydrated RBCs decreased less,this demonstrated that the biochemistry activities of the RBCs conserved better and the membrane protection needed deepgoing study in the future.