An Experimental Study On Lyophilization Of Human Red Blood Cells And Spermatozoa

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. 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 were analyzed. The freeze drying procedure of human erythrocytes and sperms were optimized.(1) Every type of cell has an osmotic tolerance limit. When the osmolality of the LPA exceeds the limit, the injury caused by the addition and removal of the LPA will be significant, even if without freeze-drying and rehydration. So it is very important to study the brittleness and the osmotic response ability. In this thesis, the procedures of the addition and removal of LPAs for human red blood cells (RBCs) were optimized firstly. Then the osmotic tolerance margin of RBCs was found with the optimized procedures of addition and removal of LPAs.KK equation was used to simulate the cell volume response during the addition and removal of permeable and impermeable agents. The discrepancy between the experiment and theory simulation results was analyzed by the specific properties of RBCs. The work of this part also supplies a basic for the analysis of the results of rehydration experiments in the followed part and the mechanism of rehydration injury.(2) The factors that affect the lyophilization of RBCs, including different permeable protective agents, polymers and freezing rate, were studied. The phase transition properties of the LPAs were measured with differential scanning calorimeter. The micro-configurations of the RBCs lyophilized with different LPAs were observed by electrical scanning microscope. The LPAs and lyophilization procedures were optimized.The discrepancy of injury caused by different permeable agents during the addition is the result of the permeability of water (Lp), permeability of protective agents (Ps) and the reflection coefficient (a). The influence of different Lp, Ps and σ on the cell volume excursion during addition and removal of LPA was analysis by KK equations. According to some values of Lp, Ps and a of the membrane of RBCs from some literatures, the results of the simulation with KK equations accorded with those of the experiments.According to the literatures, the value of Lp of RBC membranes is much bigger than that of many other cells. This is maybe one reason why the optimal cooling rate of cryopreservation or lyophilization of RBCs is bigger than many other cells. In this...