| The project is supported by the National Nature Science Foundation of China and the key Subject Foundation of Education Committee of Shanghai. In this study, the mononuclear cells were freeze-dried in aseptic condition, and residual moisture content was measured. Then the microscopic structure was evaluated and cell culture experiment was carried out for the first time.Umbilical cord blood (UCB) is now recognized as an alternative source of hematopoietic progenitor cells (HPCs) for transplantation. It has been shown to contain a similar or higher proportion of HPCs than adult bone marrow, in addition to a greater proportion of primitive progenitor cells. As cell therapies advance from research laboratories to clinical application, there is the need to transport cells across long distances and long period of time while maintaining cell viability and function. At present, cord blood cells are successfully stored and shipped under liquid nitrogen vapor currently, but this needs expensive low-temperature equipments and costs are high. The ability to store these cells in the desiccated state at ambient temperatures would provide tremendous economic and practical advantage.At present, cord blood can only be preserved at ultra-low temperatures. In former study, cord blood was tried to preserve by freeze-drying. In that study, both the mononuclear cells and the whole blood fraction of human cord blood have been freeze-dried with different protective protocols. The highest cell numerical count recovery of MNC was 75%, while that of whole blood only reached 39%. At the same time, the highest viability of recovered MNC was 98.57%, and the highest recovery of CD34+ was 59%.In this study, the aseptic operation was guaranteed during the whole experiments, which made the cell culture possible.The lyo-protective effect of FBS was evaluated by cell recovery and the residual moisture content was measured. The samples were frozen firstly by different cooling protocols. Afterwards, they were vacuum-dried at a selected shelf temperature of -30℃for 31 hours during the main drying stage, and then vacuum-dried at 15℃for the second drying stage lasting 9 hours. The residual moisture content was important to freeze-dried products. A thermal gravitative analytical balance was used to determine the residual moisture content of the samples,which was recommendable and precise. The result was 6.5±0.87% and the cell numerical recovery increased by 8%.The result of cell numerical recovery showed that fetal bovine serum had a suitable concentration as lyo-protective solution. When its concentration was higher than 10%, the cell recovery would decrease correspondingly.In order to study the proliferation ability of freeze-dried MNC, the cell culture experiments of freeze-dried cells were carried out for the first time. After 7 days, there were about 40% cells alive and many small cell colonies, but no big one was found. The culture result also showed that the aseptic operation was guaranteed during the whole experiments.The TEM micrographs were used to find the difference of internal structure of cells before and after freeze-drying, which was never reported in the world. The results showed that the rehydrated MNC were nearly uniform in size and they were smaller than the fresh ones. Cytoplasm area of rehydrated MNC looked less than the fresh MNC. |
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