Wusteman Monica, Robinson Martin, Pegg David
Medical Cryobiology Unit, Department of Biology, University of York, York YO10 5YW, UK.
Cryobiology. 2004 Apr;48(2):179-89. doi: 10.1016/j.cryobiol.2004.01.002.
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.
如果要将大块组织和器官成功低温保存，就必须找到某种方法，尽量减少传统冷冻保存方法中形成的冰对细胞外结构的破坏。使用足够高浓度的冷冻保护剂（CPA）使组织玻璃化而非冷冻，是解决这一问题的一种可能方案，并且胚胎和弹性动脉在玻璃化后功能得以保留，这表明至少一些细胞和组织能够承受该过程所需的高浓度CPA。然而，将玻璃化技术应用于大块组织和器官还存在其他问题。这些问题涉及CPA在组织中达到平衡所需的额外时间及其对毒性损伤的影响、难以实现足够快速且均匀的冷却速率以产生所需的玻璃态，以及为避免反玻璃化所需的更快且更均匀的升温速率。温度不均匀会增加快速升温过程中玻璃体内产生机械应力和破裂的风险。本文综述了克服这些问题的可能策略及已取得的进展。这将包括CPA混合物渗透到整个组织中，以及通过加入冰抑制剂和糖类等辅助剂来降低其毒性的可能性。鉴于CPA浓度的可耐受限度很可能不足以防止冷却过程中冰核的形成，介电加热对组织进行升温目前是在大块组织中实现足够快速升温速率的唯一实用方法。本文回顾了微波的生物学效应，并讨论了其在使各种形状的组织模型升温过程中产生所需均匀性方面的有效性。
