Freeze-drying of mononuclear cells and whole blood of human cord blood

The research on haematopoietic stem cells of human cord blood has become more important recently. People have concentrated on the preservation of cord blood stem cells. At present, cord blood can be preserved at ultra-low temperatures. In this study, we try to preserve cord blood and its constituents by freeze-drying. The experiments on both the mononuclear cell content and the whole blood of human cord blood were carried out respectively. The samples were frozen firstly by different cooling protocols in the presence of PVP, sucrose, and mannitol. Afterwards, they were vacuum-dried at a selected shelf temperature of -30 degree C for the main drying stage, and then vacuum-dried at 15 degree C for the second drying stage. The entire time of the freeze drying was 52 hours. Samples were stored at room temperature for 2 days prior to evaluation. Subsequently, the dried samples were suspended in an isotonic phosphate-buffered saline solution. The recovery of the cells were tested by a haemacytometer, and the highest cell numerical count recovery of MNC was 75.0 percent (SD = 4.1 percent) (P = 0.01), obtained in the protocol of 40 percent PVP + 20 percent sucrose + 10 percent Mannitol. The viability of the nucleated cells measured by PI staining and the ratio of the number of CD34+ to the number of lymphocytes (by the FITC anti-human CD34+ conjugated antibody method) were measured using a flow cytometer (FCM). The protocol of 40 percent PVP + 20 percent sucrose + 10 percent fetal bovine serum had the highest viability of 98.6 percent (SD = 0.7 percent) (P = 0.01). The highest ratio of CD34+ to lymphocytes was 1.2%, and the highest recovery of CD34+ was 68.4 percent (SD = 39.5 percent) (P = 0.05). Comparing the results of the lyophilized MNC subfraction with that of the whole blood, the lyophilization of the isolated MNC was more successful than that of whole blood.