Olga Kukal & Thomas Allen

During the period from 2006 until 2018 the authors undertook a series of studies to determine the benefits of cold shock inhibitors, cryoprotectants, compounds that promote desiccation resistance, and increase membrane plasticity during cooling. Using compounds that are produced by arthropods prior to and during overwintering in the high arctic comparative studies were conducted on two model mammalian systems.

We selected bovine spermatozoa because is its membranes are rich in saturated fatty acids and remain flexible during cooling. These haploid cells are regularly cooled from 37°C to 15°C in a water jacket during the first hour (0.36°C/minute), then removed from the water jacket at cooled from 15°C to 4°C during a second hour (0.183C/minute). The post cooling survival in a milk or egg yolk based diluent containing 8% glycerol is in excess of 90%. These rates of cooling are considerably slower than the 1°C cooling rate normally used to cool and freeze cells for research and clinical use. Bovine Spermatozoa has been frozen in the dairy industry for over 50 years and sires have been selected during that time for their ability to freeze and survive. In this best of all possible cases the post thaw survival is rarely 70% and most of the survivors succumb to cryo-injury during the next 18 hours.

The germ cells we used for comparison were porcine spermatozoa because their cell membranes are cholesterol deficient containing a large percentage of unsaturated fatty acids. Porcine spermatozoa’s cell membranes become brittle and fracture during cooling. This property requires the pork industry to cool aliquots for insemination no colder than 16°C in order to prevent excessive cell mortality. Diluted ejaculates are cooled from 37°C to 16°C over 4 hours (0.0875°C/minute) considerable slower that bovine spermatozoa or mammalian cells for research and clinical purposes.

Extensive studies were conducted on each of the model system exploring the efficacy of the novel compounds used by arctic arthropods. The formulations included potent buffers to limit acidosis but did not include raw egg yolk, milk or serum albumin. It is interesting to note that media for porcine cells performed poorly when used on bovine germ cells and the most effective formulation for preserving bovine spermatozoa was not the most beneficial to porcine cells.

In exploration of Olga’s Central Hypothesis we cooled isosmotic solutions containing each cell type and stored them at -2°C. Varying the cooling rate for each cell type we determined that when an appropriate media formulation and cooling protocol is used the cells can be successfully cooled and stored. Porcine cells can be cooled to -2° over a 12 hour period (0.054°C/minute) and then survive for 7 days with only ~10% mortality. When bovine cells were cooled using the industry standard protocol (above) for -4°C extended to -2°C they can also be stored for 7 days with only ~10% mortality.

The success of these studies led us to conclude that most mammalian tissue can be cooled to high sub-zero temperature without freezing extending its viable shelf life over the current art of storage at +4°C.