[Cryogenic preservation of erythrocytes by a droplet freezing technic]

The droplet freezing technique has two major advantages: firstly the good recovery of frozen erythrocytes with low concentration of cryoprotectants, and secondly the simplification of the post-thaw treatment. However, this technique has not been utilized for blood preservation for transfusion due to difficulties of the sterile freeze-thawing operation. In this study, the technique was modified to perform the freeze-thawing in a closed system under sterile conditions. An apparatus with a new type of nozzle was devised for this purpose. Fifty milliliters of packed red blood cells mixed with an equal volume of additive solutions were frozen in liquid nitrogen in droplet form (0.7 mm in diameter). The frozen droplets were collected into an aluminium container (18 X 18 X 0.5 cm) and thawed by immersing the container into a water bath at 47 degrees C. After thawing, the red cells were washed with either normal saline or hypertonic sugar solutions and resuspended in normal saline. Although the washing procedure involving centrifugation was necessary at least once to remove free hemoglobin and fragile cells produced during freeze-thawing, the post-thaw treatment of the modified droplet freezing was much more simple than that of ordinary bulk freezing technique. Approximately 85% of the frozen cells were recovered with cryoprotective additive solutions consisting of 0.4-0.5 M sugar (maltose or glucose) and 0.4-0.5M glycerol in 0.03 M sodium chloride. The osmotic fragility and ATP level of the post-thawed red cells were little changed in comparison with that of unfrozen cells. These results suggests a possibility of this technique for practical application in the future.