Kirichek Oleg, Soper Alan, Dzyuba Boris, Callear Sam, Fuller Barry
ISIS facility, STFC, Rutherford Appleton Laboratory, Harwell Oxford Campus, Didcot, Oxon, United Kingdom.
South Bohemian Research Center of Aquaculture and Hydrocenoses, University of South Bohemia in Ceske Budejovice, Zatisi, Vodnany, Czech Republic.
PLoS One. 2015 Mar 27;10(3):e0120611. doi: 10.1371/journal.pone.0120611. eCollection 2015.
The nucleation and growth of crystalline ice during cooling, and further crystallization processes during re-warming are considered to be key processes determining the success of low temperature storage of biological objects, as used in medical, agricultural and nature conservation applications. To avoid these problems a method, termed vitrification, is being developed to inhibit ice formation by use of high concentration of cryoprotectants and ultra-rapid cooling, but this is only successful across a limited number of biological objects and in small volume applications. This study explores physical processes of ice crystal formation in a model cryoprotective solution used previously in trials on vitrification of complex biological systems, to improve our understanding of the process and identify limiting biophysical factors. Here we present results of neutron scattering experiments which show that even if ice crystal formation has been suppressed during quench cooling, the water molecules, mobilised during warming, can crystallise as detectable ice. The crystallisation happens right after melting of the glass phase formed during quench cooling, whilst the sample is still transiting deep cryogenic temperatures. We also observe strong water isotope effects on ice crystallisation processes in the cryoprotectant mixture. In the neutron scattering experiment with a fully protiated water component, we observe ready crystallisation occurring just after the glass melting transition. On the contrary with a fully deuteriated water component, the process of crystallisation is either completely or substantially supressed. This behaviour might be explained by nuclear quantum effects in water. The strong isotope effect, observed here, may play an important role in development of new cryopreservation strategies.
在冷却过程中结晶冰的成核和生长，以及复温过程中的进一步结晶过程，被认为是决定生物物体低温保存成功与否的关键过程，这在医学、农业和自然保护应用中均有涉及。为避免这些问题，一种称为玻璃化的方法正在被开发，该方法通过使用高浓度的冷冻保护剂和超快速冷却来抑制冰的形成，但这仅在有限数量的生物物体和小体积应用中取得了成功。本研究探索了一种先前在复杂生物系统玻璃化试验中使用的模型冷冻保护溶液中冰晶形成的物理过程，以增进我们对该过程的理解并确定限制生物物理因素。在此，我们展示了中子散射实验的结果，这些结果表明，即使在骤冷冷却过程中冰晶形成已被抑制，但在复温过程中被激活的水分子仍可结晶形成可检测到的冰。结晶发生在骤冷冷却过程中形成的玻璃相熔化之后，此时样品仍处于深低温状态。我们还观察到水同位素对冷冻保护剂混合物中冰晶形成过程有强烈影响。在使用完全由氢原子组成的水成分进行的中子散射实验中，我们观察到在玻璃熔化转变后立即发生了结晶。相反，在使用完全由氘原子组成的水成分的实验中，结晶过程要么完全被抑制，要么被显著抑制。这种行为可能由水中的核量子效应来解释。此处观察到的强烈同位素效应可能在新的冷冻保存策略的开发中发挥重要作用。
