Study on the Freeze-drying Preservation of Large Volume of Human Platelet
|Course||Refrigeration and Cryogenic Engineering|
|Keywords||Human platelet Freeze-drying Large volume Water vapor diffusioncoefficient Primary drying temperature|
The existing method of platelet preservation has several limitations. Freeze-drying provides a potentially ideal alternative for a long time preservation of human platelets which has a great deal of merits, such as long guarantee period, easy achieved preservation conditions, portability of the products, direct usage after rehydration and so on. The research on the lyophilization of platelets has made encouraging progress in recent years. In the author’s research group, small samples (1-2mL) of lyophilized human platelets can reach a relatively high survival and recovery rates, whereas there still exists a big gap between the clinical application and research. The aim of this thesis is to understand the primary drying process in depth and to scale up the volume of lyophilized human platelets samples. The following work has been carried out:(1) Through experiments, the water vapor diffusion coefficient in the dried part of the lyophilizing sample was measured under the following operation conditions: vacuum at6Pa, pre-freezing temperature at-60℃, pre-freezing velocity at about10K/min, primary drying temperature at-30℃. Its value was fitted to be1.6589×10-5m2/s.(2) The COMSOL Multiphysics software was adopted for the simulation of the primary drying process in glass vial. The results showed that the primary drying process of platelet suspension was mass transfer constrained and the existence of the vial bottom curve had great influence on the drying speed. The effects of the initial sample height and the shelf temperature to the drying speed were also studied. (3) The influence of the shelf temperature during the primary drying process on the lyophilized human platelets was investigated. The cell recovery of rehydrated freeze-dried platelets, mean platelet volume (MPV) and platelet distribution width (PDW) were evaluated respectively. The results showed that when the shelf temperature was at-30℃, the cell recovery rate after rehydration reached89.0±4.8%, the MPV and PDW were12.4±1.4fL and18.3±1.4%, respectively, which were both within acceptable ranges. The morphology of freeze-dried platelets remained integral and there were no visible flaws. The non-apoptosis rate of rehydrated platelets was87.5±2.5%and the non-activation rate was14.6±5.9%, which were detected by flow cytometry.(4) A preliminary research was carried out with a specially designed blood vessel for the freeze-drying of large volume (100mL) of human platelets. The recovery rate after rehydration reached66.3±2.1%. Compared with fresh control, the MPV and PDW values increased a little, to12.4±0.6fL and18.0±0.7%respectively, but they were still in normal ranges. The non-apoptosis rate was about77.8±6.3%. The non-activation rate was8.2±0.9%, which means most rehydrated platelets lost their viability. The aggregation test showed that the aggregation function of rehydrated platelets was weakened to approximately50%of fresh platelets.