Development of a Directional Solidification Device for Cell Cryopreservation

Abstract

The objective of this study was to develop a reliable, low-cost, and practical directional solidification device (DSD) for cryopreservation of biological cells and tissues. In this study, a linear temperature distribution was established in an aluminum block between a higher temperature source (T_h = -5 to 30°C) and a lower temperature sink (T_c = -60 to -85°C). Liquid nitrogen vapor was used to maintain T_c. Two cartridge-heaters were used to maintain T_h. Various cryopreservation media (with glycerol, ranging from 0 to 4 M) were placed in long-cylindrical plastic straws (0.5 mL in volume, 2 mm in outer diameter, 150 mm in length, 0.02 mm in wall thickness). The straws were pushed (through a cylindrical channel in the block) from the warm end of the block (T_h) to the cold end (T_c) at a constant speed to achieve a corresponding constant cooling rate (up to -200°C/min depending on the speed and the source/sink temperatures). Thermocouples were used to measure/monitor temperature distributions in both the block and the sample inside the straw. After the validation of the DSD, using the DSD and a commercial cooling machine, respectively, human sperm samples in TALP solution with 1 M glycerol were cooled at - 10°C/min to -85°C, and then dropped into liquid nitrogen ( -196°C). After 30 min in liquid nitrogen, the straws with sperm samples were warmed in a stirred water bath at 22°C. Motility of the sperm in each sample was measured before and after freezing-thawing processes. It was found that a cooling rate up to -200°C/min can be achieved by changing the moving speed of the straws in the DSD or the source/sink temperatures. There was no significant effect of glycerol concentration on the cooling rates (p < 0.001). Cryosurvival rate of human sperm using the DSD agreed very well with that using the commercial cooling machine. The present DSD was shown to be a useful tool to achieve a large range of controlled cooling/warming rate, and appeared to be a reliable and inexpensive cooling/warming device for indoor and outdoor applications.