Forced-convective vitrification with liquid cryogens

Cell cryopreservation by vitrification generally requires using vitrification solutions with high concentrations of cryoprotectants (CPAs), which are toxic and induce osmotic stresses associated with the addition and removal of CPAs. To increase the cooling rate and reduce the CPA concentration required for vitrification, this study proposed an innovative approach, named forced-convective vitrification with liquid cryogens, in which liquid oxygen at a temperature below its boiling point (LOXbbp) was used as the cryogen to reduce the generation of insulating bubbles of gaseous oxygen and the sample was subjected to a constant velocity to remove insulation bubbles from the sample. Results show that changing the cryogen from liquid nitrogen at its boiling temperature (LNabp) to LOXbbp, increasing the sample velocity and reducing the test solution volume increased the cooling rate and thereby decreased the CPA concentration required for vitrification. Using the same velocity (1.2 m/s), the cooling rate achieved with LOXbbp was 2.3-fold greater than that achieved with LNabp. With LOXbbp, the increase in the sample velocity from 0.2 to 1.2 m/s enhanced the cooling rate by 1.9 times. With LOXbbp, a velocity of 1.2 m/s and a test solution volume of 1.73 μl, the CPA concentration required for vitrification decreased to 25%. These results indicate that the new approach described here can reduce the CPA concentration required for vitrification, and thus decreases the toxicity and osmotic stresses associated with adding and removing the CPA.