A highly active mineral-based ice nucleating agent supports in situ cell cryopreservation in a high throughput format.

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Freezing injury: the special case of the sperm cell.

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice although no convincing evidence of intracellular ice formation has ever been obtained. We demonstrate that the high intracellular protein content together with the osmotic shrinkage associated with extracellular ice formation leads to intracellular vitrification of spermatozoa during cooling. At rapid rates of cooling the cell damage to spermatozoa is a result of an osmotic imbalance encountered during thawing, not intracellular ice formation. The osmotic imbalance occurs at rapid cooling rates due to a diffusion limited ice crystallisation in the extracellular fluid, i.e. the amount of ice forming during the cooling is less than expected from the equilibrium phase diagram. This explanation allows insights into other aspects of the cryobiology of spermatozoa and it is anticipated that this understanding will lead to specific improved methods of conventional cryopreservation for mammalian spermatozoa. It is also likely that this model will be relevant to the development of novel technologies for sperm preservation including vitrification and freeze drying.