12. From ice in the veins, through unfrozen fish and frozen frogs to isochoric preservation, ad Astra

Abstract

Star Wars V: The Empire Strikes Back – One of the most dramatic scenes in the movie is when the Dark Lord successfully entombs Han Solo in carbonite in a state of suspended animation for transportation to Tatooine to receive a bounty from Jabba. Recently records show that the film has made so far $819,808,300. I doubt that the entire scientific community, together, through the last century, has received from society, 1% of this amount to carry out research on cryobiology. Imagine the state of science in cryobiology if society would have invested 10% of the amount, this one movie grossed, in research. This 6 min presentation will give a brief review of the research in my lab on cryobiology and evidence on why cryobiology could work. Cells survive freezing to cryogenic temperature while organs do not. To understand why, we build a directional solidification stage that can mimic the process of freezing in bulk tissue under a microscope (Rubinsky and Ikeda, 1985). The research has shown, that because of geometrical constrains unique to tissue and absent in single cells, freezing causes damage to the extracellular matrix of the tissue (Rubinsky and Pegg, 1988). Avoiding this mechanism of damage should be an important target in designing cryopreservation protocols. A way to avoid this damage is by finding ways to reduce temperature without formation of ice or by forcing the ice to form at sites where it will not cause mechanical damage. This is what nature does. Arctic and Antarctic fish produce chemicals that allow them to live without ice in their system at temperatures below the normal freezing temperature of physiological saline (Devries, 1971). Using a combination of antifreeze proteins for their cell membrane protection properties (Rubinsky, Arav, et al., 1991) and freezing depression properties (Rubinsky and Devries, 1989) and glycerol for the freezing depression properties we successfully cryopreserved a liver at subzero temperatures (Ishine, Rubinsky, et al., 2000) and transplanted the liver in an animal that lived normally, following the transplantation. Rana Sylvatica frogs survive freezing by modulating the freezing process with glucose, in such a way that the tissue extracellular matrix is not impaired (Storey, Bischof, et al., 1992) and thawing is achieved in an optimal manner for a living animal (Rubinsky, Wong, et al., 1994). Combining the knowledge from Antarctic fish and freeze tolerant frogs, we successfully preserved aa heart for 24 h and transplanted the organ (Amir, Rubinsky, et al., 2004). One way to reach high subzero temperatures without freezing a cooled organ is by isochoric subcooling (Rubinsky, Perez, et al., 2005). Unpublished results on long-term preservation of hearts with this method will be shown.