59. High concentration ice nucleation for controlled rate freezing of complex biological systems

Abstract

Cryopreservation of human organs would facilitate long term organ banking and potentially go a long way to remedying the global shortage of organs for transplantation. To date, almost all attempts at cryopreservation of whole organs have used vitrification methods. Controlled rate freezing, which is commonly used for freezing of various types of cell suspensions, is unlikely to work well for whole organs as ice crystals will inevitably nucleate and grow to damaging sizes. Here, we propose an alternative to vitrification for the cryopreservation of whole organs. By introducing very large numbers of small ice nucleating particles to an organ it might be possible to induce formation of very large numbers of small, relatively harmless crystals, rather than small numbers of large, and therefore damaging ice crystals. The ice nucleating particles used would have to be less than 50 nm in diameter, and would have to nucleate ice at a high and very uniform temperature. The minimum known size of effective ice nucleating particles has been falling rapidly of late, meaning that particles of the requisite size and effectiveness are now known, although it is likely that further work will have to be conducted to generate sufficiently uniform ice nucleation temperatures. Uniformity of crystal size is of vital importance as larger ice crystals are more thermodynamically stable than smaller ones and as such will tend to grow at the expense of smaller crystals, rapidly producing ice of a damaging size. To achieve this, an organ would need to be supercooled very uniformly so that nucleation occurred at a similar time throughout. Once extracellular nucleation had been triggered the careful control of temperature would be needed to ensure optimal dehydration of cells for successful cryopreservation. It is anticipated that the size of crystals can be tuned by varying the concentration of ice nucleating particles. On rewarming, a uniform ice crystal size will prevent excessive recrystallization of large ice crystals and the ensuing tissue damage that is often observed during vitrification procedures. Overall, it might be possible to use a controlled rate freezing type method with larger, more complex systems than is currently possible. Although such an approach provides numerous potential benefits there are many uncertainties that will need to be explored through experiment. It is proposed that such experiments could start with light microscopy of dyed cryoprotectant solutions to determine if ice crystals of suitably small size can indeed be formed before moving onto cell suspensions, tissues and whole organs.