Vitrification of large tissues with dielectric warming: biological problems and some approaches to their solution

If large pieces of tissue and organs are to be successfully stored at low temperatures, some means must be found to minimize the disruption of extracellular structures by the ice that develops during conventional cryopreservation methods. The use of sufficiently high concentrations of cryoprotectant (CPA) to vitrify rather than freeze the tissue is a possible solution to this problem, and the retention of function of embryos and elastic arteries after vitrification suggests that some cells and tissues at least can withstand exposure to the high concentrations of CPA necessary for this process to occur. There are, however, additional problems in applying vitrifying techniques to bulky tissues and organs. These are related to the additional time required for tissue equilibration of CPA to occur and the consequences for toxic injury, the difficulty in achieving sufficiently rapid and uniform cooling rates to produce the required glassy state, and the even more rapid and uniform warming rates that are necessary to avoid devitrification. Non-uniformity of temperature will increase the risk of mechanical stresses and fractures developing in the glass during rapid warming. This paper reviews possible strategies and the progress that has been made in overcoming these problems. This will include the permeation of CPA mixtures into whole tissues and possibilities for reducing their toxicity by the inclusion of adjuncts such as ice inhibitors and sugars. The warming of tissues by dielectric heating is currently the only practical means by which sufficiently rapid rates can be achieved in bulky tissues given that the tolerable limits of CPA concentration will most likely be insufficient to prevent the development of ice nuclei during cooling. The biological effects of microwaves are reviewed and their effectiveness in producing the required uniformity in warming of tissue models of various shapes are discussed.