| As freeze drying (lyophilization) preservation has more significant virtues than many other long-term preservation methods, it has become one new research concentration of long-term preservation of cells. The factors that influence the recovery rate of the freeze dried cells includes: 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. All the work in this thesis will be explored around the topic of the injure mechanism of lyophilization. With many experiments, the factors which affect the recovery rate of freeze dried cells and the injury mechanism are analyzed. The freeze drying procedure of human erythrocytes are optimized. The factors that affect the lyophilization of RBCs, including different permeable protective agents, polymers and freezing rate, are studied. The injury during rehydration is another very important reason that causes low recovery rate of lyophilized RBCs. In this thesis, the influences of different rehydration solutions, temperatures and velocities are studied. The lyophilized RBCs are rehydrated by the vapor in the environment scanning electrical microscope (ESEM) for the first time. The volume changes of the cells are measured. The experiment and the discussion about the injury caused by the addition and removal of the lyo-protective agents can explain the reason why the pre-rehydration can increase the recovery rates. This experiment also discloses the injury mechanism of rehydration caused by the cell volume response and excursion during that process.Objective The objectives of this experiment were to explore the factors which affect the lyophilization of RBCs, including different permeable protective agents, polymers and freezing rate, to study the protection mechainism of the wapor pre-rehydration towarding lyophilizing RBCs, and to analyse the injury of the hemolysis by the osmotic changes and the volume changs of the cells, so as to optimize the rehydration program and reduce the hemolysis rate. Methods Healthy donor's blood gotten from Hefei Red Cross Blood Center was re-suspended in osmotic NaCl solution, then was centrifuged for 3 min at the rate of 690 g . The upper buffer and white cells were removed. Then the cells were re-suspended and washed again. The cells were washed for 3 times. At last the PCV(Packed cell volume, PCV)of the red blood cell suspension was 80%-83%. The RBCs were incubated in 30.27%(W/V)trehalose solution at 37℃for 3 hours , and the different lyo-protective agents were added to the cells by 4 steps with 3 min interval between each step. The red blood cell suspension with protective agent was dispatched to many vials, and each vial has 250μL suspension. These vials were put into the shelf which had been set in just program of freeze-dryer. After that, vials were taken out and were set into the icebox of 4℃. 4 weeks later, the vials were taken out of the icebox , the vials and rehydrationed solutions were also warmed-up to 37℃. 1mL rehydration solution was put into every vial by 4 steps (every step's capacity was 0.25ml). Then, the vails were shaken and blended well-proportioned, every step's interval was 3 min. PBS was put into the vails by 4 steps of 290μl,540μl,1310μl and 7860μl until they have nature osmotic. The dilute samples were determined their gross concentrations of Hb. Samples was centrifugaled at 110 g for 10min, and were determined their superstratum concentrations of Hb. The recovery rate =[( samples'gross concentration - superstratum concentration )/ samples'gross concentration]×100%. The several groups with different lyo-protective agents, rehydration temperatures, cooling rates or rehydration rates were evaluted the morphology, the hemolysis rate, the ATP level , the 2,3-DPG level and so on. Data presented are mean±SD and statistical differences between two groups were analyzed using Student's t-Test.Results1. The lyo-protective agents composed of 0.8 M glycerol(or DMSO,or glucose, or EG,or 1,2-PG)were dissolved in 1/4 PBS. The recovery rates of freeze-dried and rehydrated cells were 43.1%±3.3%;35.3%±2.1%;45.2%±2.5%;43.9%±2.8%;42.7%±3.6%. The recovery rate of 1,2-PG group is higher than others (significantly different, P<0.01).2. Studied from differential scanning calorimeter (DSC), the lyo-protective agent's (composed of PVP+trehalose+glycerol+BSA ) crystal temperature was -13.2℃,vitrification temperature was -38.9℃. The cooling temperature of the materials in the vials should be lower than -40℃. According to the diathermancy difference in temperature between the shelf and the materials (about 15℃in low temperature) , set the shelf cooled to -70℃and let the materials stay some time in this temperature. The shelf's temperature of first drying program was -60℃.3. The materials in the vials were cooled with 3 manner: the vials were put onto the shelf in room temperature, then the vials and the shelf were cooled together until the temperature get -70℃; the vials were put onto the shelf which had been cooled to -70℃; the vials were put into liquid nitrogen for 30 s, then were put onto the shelf which had been cooled to -70℃, and then were freeze-dried. The recovery rates of freeze-dried and rehydrated cells were 40.8%±2.3%;49.5%±1.9%;56.3%±1.7% (significantly higher, P<0.01).4. The freeze-dried samples were rehydrationed with rehydration 5 kinds of solutions: PBS; PBS+10%trehalose(dissolved in distilled water)with the same volume; 10%trehalose(dissolved in distilled water); 15%trehalose(dissolved in distilled water); 15%PVP(dissolved in PBS) . The recovery rates of freeze-dried and rehydrated cells were 38.2%±3.3% ; 40.4%±5.1% ; 44.0%±4.2% ; 42.1%±4.5% ; 58.5%±4.6% respectly(significantly higher, P<0.01).5. The samples and rehydration solution were put in water bath with different temperatures(0℃,10℃,25℃,37℃,42℃). the recovery rates increase with the increase of the rehydration temperatures. The maximal recovery rates was got at 37℃(53.5%±3.0%, significantly different from other temperatures, P<0.01.). The recovery rate of RBCs at 42℃(49.3%±1.4%) is lower than that at 37℃(significantly different, P <0.01.), but higher than other temperatures (significantly different, P<0.01.).6. The lyophilizing RBCs put in cultivating box at 37℃for 3 hours were rehydated and the RBCs without pre-rehydatrion were used as control. The hemolysis rate of pre-rehydatrioned group was 22.2%±2.1%, while the hemolysis rate of the control group was 35.4%±2.4%(P <0.05); The level of 2,3-DPG in RBCs of pre-rehydatrion group was (7.01±1.17)μmol/gHb, which of the control group was (6.82±2.10)μmol/gHb(P>0.05);The level of ATP in RBCs of pre-rehydatrion group was (5.13±0.52)μmol/gHb ,which of the control group was (5.22±0.10)μmol/gHb(P >0.05);compared with control group, the recovery of packed cell volume(PCV) and the mean corpuscular volume(MCV) of pre-rehydatrioned group was significantly higher(P<0.05),and the RDW was significantly lower(P<0.05).Conclusions1. The choice of the lyo-protective agents: glycerol (15%PVP+4%trehalose+3%glycerol+5%BSA+0.05%HSI )can protect RBCs better.2. The choice of the freeze-dried programe : the vials set in the liquid nitrogen for 30s are put onto the shelf which has been cooled to -70℃for 10min, and then are freeze-dried. The cooling rate between room temperature and 0℃is -60℃/min, between 0℃and -6℃is -13℃/min, and between -6℃and -40℃is -42℃/min. The drying program is that: the materials are dried for 3 h under -60℃,-50℃,-40℃and -30℃respectively, and then dried for 2h under -20℃,-10℃,0℃,and 10℃respectively. The vacuum pressure in the chamber is 13.3 Pa. At last, the residual moisture of the dried products is about 3%～4%.3. The choice of the condition of the rehydration: rehydration solutions composed of 15%PVP (based on PBS) can enhance the recovery of lyophilizing RBCs; It is beneficial to rehydrate the freeze-dried red blood cells in 37℃and slowly. The pre-rehydatrion can maintain the integrity of RBC membrane, and significantly enhance the recovery of lyophilizing RBCs.
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