24. Diffusion limited ice crystallisation + a comment on liquidus tracking

Diffusion limited ice crystallisation. During conventional cryopreservation many of the changes which occur in a CPA solution following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of ionic species, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature. In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a nonlinear manner with temperature. We have measured the viscosity of the residual unfrozen solution that cells are exposed to during conventional freezing, and the effect of high viscosities encountered on the diffusion of water at a constant temperature during freezing and during cooling at different linear rates has been estimated. At rates of cooling faster than 100°Cmin−1 the diffusion distance during freezing was calculated to be less than 15 μ m. Validation of the diffusion calculations were confirmed by examination of the ultrastructure of the freeze concentrated matrix in samples prepared at a range of cooling rates. At a critical rate of cooling, water diffusion becomes limited by the high viscosity and the composition of the freeze concentrated matrix deviates from that of the equilibrium phase diagram, the effects on cell viability of this non equilibrium state are demonstrated. Liquidus tracking. In 1965 Farrant proposed the “liquidus tracking” method of preservation in which cryoprotectant is added progressively during cooling so that samples remain on or above the liquidus line of the equilibrium phase diagram. Low temperatures protect against cryoprotectant toxicity at high concentrations. This method has also been termed the equilibrium approach to vitrification. However due to practical issues associated with high viscosities and mixing at low temperatures the method was largely abandoned. Recently there has been revived interest in the technique as applied to the preservation of cartilage and encapsulated hepatocytes and also in ultrastructural studies where the method is known as progressive lowering of temperature (PLT). Planer products have modified their controlled rate freezer to deliver the process for cartilage. Asymptote are developing equipment for this process which will allow preservation of clinical biopsies with native immunology which can be immunolabelled and from which viable cells may be isolated for adoptive cell therapies and personalised medicine.

扩散限制的冰晶形成 在常规低温保存过程中，冰晶成核后CPA（低温保护剂）溶液中发生的许多变化与温度并非呈线性关系。诸如冰的比例、离子种类浓度、渗透压、pH值、粘度和气体溶解度等参数都随温度呈非线性变化。此外，决定细胞对冷冻反应的生物物理特性，例如细胞对水的通透性，也随温度呈非线性变化。 我们测量了细胞在常规冷冻过程中所接触的剩余未冻溶液的粘度，并估算了在冷冻过程中恒温下以及以不同线性速率冷却时遇到的高粘度对水扩散的影响。在冷却速率快于100°C/min时，计算得出冷冻过程中的扩散距离小于15μm。通过对在一系列冷却速率下制备的样品中冷冻浓缩基质的超微结构进行检查，证实了扩散计算的有效性。在一个临界冷却速率下，水的扩散因高粘度而受到限制，冷冻浓缩基质的组成偏离平衡相图，这种非平衡状态对细胞活力的影响得到了证明。 液相线追踪 1965年，法伦特提出了“液相线追踪”保存方法，即在冷却过程中逐步添加低温保护剂，使样品保持在平衡相图的液相线或其上方。低温可防止高浓度低温保护剂的毒性。这种方法也被称为玻璃化的平衡方法。然而，由于高粘度以及在低温下混合等实际问题，该方法在很大程度上被放弃了。最近，人们对该技术在软骨和包封肝细胞保存方面以及在超微结构研究（该方法在其中被称为逐步降温法，即PLT）中的应用重新产生了兴趣。Planer公司对其程控速率冷冻机进行了改进，以实现软骨的这种处理过程。Asymptote公司正在开发用于该过程的设备，这将允许保存具有天然免疫学特性的临床活检样本，这些样本可以进行免疫标记，并且可以从中分离出活细胞用于过继性细胞疗法和个性化医疗。