21. A lesson from Nature for controlling ice during organ cryopreservation by vitrification

Banking of organs using current tissue banking practices employing conventional cryopreservation by freezing is not feasible due to the well known damage caused by intra and extra-cellular ice formation. An alternative cryopreservation approach is known as vitrification. There has been considerable success in ice-free vitrification of small tissue samples but better ice control is required for samples > 10 mL in volume. Development of novel cryoprotectants and methods that will facilitate clinically effective banking by vitrification of large vascularized tissues and organs is needed. There are lessons to be learned for organ vitrification from Nature. Freeze avoiding insects such as Cujucus clavipes beetle larvae from the interior of Alaska vitrify and survive temperatures that routinely reach −60 °C with some individuals surviving exposure to −100 °C. At the onset of winter these insects combine changes in behavior with increases in cryoprotectant content (such as glycerol, proline, trehalose), dehydration, production of antifreeze compounds (proteins and glycolipids), and diapause. The cryoprotectant concentration can achieve 4.2 M, approximately half the concentration required for ice-free cryopreservation of tissue and organ therapy products. Recombinant copies of insect-derived antifreeze proteins are available, so they can be manufactured, and they are up to 10 times more effective in ice modulation (thermal hysteresis) than the well known fish-derived antifreeze proteins that have proven ineffective or detrimental for cell cryopreservation. Recently we have discovered that certain insect-derived antifreeze proteins significantly increase cell viability during cryopreservation by freezing. Similarly, in unpublished studies we have found that an insect-derived antifreeze glycolipid, with thermal hysteresis activity similar to that of hyperactive insect antifreeze proteins, can significantly increase cell viability post-cryopreservation using freezing methods. These glycolipids have been found in several species including freeze tolerant and avoiding insects, freeze tolerant frogs, and freeze tolerant plants. Unlike the antifreeze proteins, which are diverse in structure across species, these glycolipids are nearly identical. While we anticipate that eventually it will be possible to synthesize the saccharide component (s1-4 linked xylose-mannose), at present the antifreeze glycolipid is harvested from natural sources, especially plants. Translation of this lesson from Nature requires a better understanding of how animals prepare themselves for diapause and hibernation so that we can reproduce these phenomena in organ donors in vivo, under physiological conditions post-mortem prior to organ procurement, and/or organs ex-vivo before cryopreservation in cryoprotectant formulations that incorporate antifreeze compounds. It is anticipated that utilization of antifreeze proteins and glycolipids will enable utilization of less cytotoxic cryoprotectant formulations with enhanced ice control during cooling and warming of larger biological structures than currently possible. In the big picture it is likely that they will be combined with other strategies presented in this session on controlling ice formation including improved cryoprotectant addition and removal methods, pressure assisted cooling, stress relief by annealing, synthetic antifreeze compounds, and nanoheater assisted rewarming. Once we have ice formation under control we must be concerned with ischemic reperfusion injury, the final hurdle upon in vivo transplantation in patients.