Study On The Optimization Of Freeze-drying Protocol For Human Platelets

Freeze-drying is an ideal alternative for long-term preservation of human platelets. Lyophilized platelets can be stored at room temperature for a long time stably, and can be conveniently transported. And they can recover the viability immediately after rehydration. Since platelets may encounter the damages caused by frozen shock, activation damage, desiccation and the rehydration stress, the lyophilization technique of platelets is difficult and still far away from the clinical application. The aim of this thesis is to optimize the freeze-drying and rehydration protocol so that the morphology and the viability recovery of the rehydrated platelets can be improved. The following studies were carried out:The feasibility of using the ultrasound to facilitate the loading of trehalose into platelets was studied. Based on the technique of loading trehalose into human platelets through the fluid-phase endocytosis at 37℃, the addition of low frequency ultrasound is advantageous to the process. After being radiated by 25 kHz,0.4 W/cm2 ultrasound for 1 h, the average amount of trehalose loaded into platelets increased by 1.46 times, compared with the traditional incubation method. Moreover, the platelets being radiated had no distinct statistical differences with the fresh platelets, by the haematology analysis, morphology observation and flow cytometry analysis.The effects of the annealing treatment to freeze-dried platelets were studied. An orthogonal experiment was conducted, including the factors of the cooling rate (A), the annealing rate (B), the annealing temperature (C), and the annealing time (D). The numerical cell recovery rate, cell morphology, and activation rate of the rehydrated freeze-dried platelets were considered. The results showed that, these four parameters of pre-freezing process all had significant impacts on the numerical cell recovery rate when the other parameters of the freeze-drying process were fixed, the order of influence degree was:B>C>D>A. The optimal protocol was as follows. First, the human platelet suspension was frozen to below -40℃at a rate of 20℃/min, maintained for 2 h, then the shelf temperature was shifted to -30℃at a rate of 1.5℃/min and kept for 0.5 h. The rehydrated platelets had an integral ultrastructure under the electron microscope.The effects of the rehydration solution composition, the prehydration time and temperature were studied. For the solution of four kinds of plasma concentration, the solution of 75% plasma can be taken as the best rehydration solution, the cell recovery of the freeze-dried platelets after rehydration reached (89±3.6)%, the increasing percentage of PDW was the least. The prehydration tests of the freeze-dried specimens of 1 mL and 2 mL were conducted. As a result, the prehydration temperature of 35℃and 37℃were almost the same. For the specimens of 1 mL, prehydration for 15 min at 37℃, and for the specimens of 2 mL, prehydration for 90 min, were the optimal prehydration duration, the final moisture contents were almost the same.Based on the optimization studies of the protocol above, the viabilities of the freeze-dried human platelets were further evaluated. The protocol was as follows. Platelets suspension of 1 mL after trehalose loading, were frozen to -60℃at a rate of 20℃/min, then primarily dried at-40℃, and the secondarily dried at 20℃. After being stored for 30 days, the freeze-dried platelets were prehydrated in saturated vapor with partial pressure of 5.9 kPa for 15 min at 37℃, then rehydrated in 75% plasma solution. The morphology of the platelets remained integral. After being stimulated by 1 U/mL thrombin, the maximal aggregation rate of the platelets reached 82.8% of that of the fresh human platelets, and the aggregation slope in 4 minutes reached 15.75%/min, was 16.9% of that of fresh platelets.The ultrasound-aiding method put forward in this thesis for loading trehalose into platelets provides a new approach for loading lyoprotectants into mammalian cells. The optimized protocol could be valuable to the freeze-drying of large volume of human platelets, and their clinical applications.