Stochastic-Process Modeling of Intracellular Ice Formation during Freezing of Biological Cells

活细胞冷冻保存方案的设计需要确定冷冻保护剂的类型和浓度、冷却速度和最终预冷温度，以防止细胞内结冰。在这个项目中，将采取一种全新的方法，包括开发细胞冷冻过程中细胞内结冰(IIF)发生率的通用概率模型。将在哺乳动物种质系统上进行详细的冷冻显微镜实验，以(1)确定细胞过冷耐受性的存在和大小，(2)建立瞬时和随时间变化的IIF概率模型的数学形式，以及(3)确定低温保护剂对过冷耐受能力和IIF概率的参数影响。可靠性理论的概念将被用来开发IIF概率的数学模型，并综合细胞冻结行为中的各种随机性来源。将在植物细胞系统上进行一组简化的实验，以测试建模方法的普遍性。该模型将用于探索创新的冷却方案的可行性，这些方案可以显著提高细胞存活率。该项目将在发展细胞对冷冻的反应的工程模型方面向前迈出根本的一步，并创造用于设计冷冻保存方案的新的分析工具。该模型的开发将有广泛的冷冻应用：人和动物的精子、卵母细胞和胚胎；器官和专门组织；无性繁殖物种的植物茎尖；以及濒危动植物种质的保护。

The design of protocols for cryostorage of living cells requires the determination of cryoprotectant type and concentration, cooling rate, and final prequench temperature that prevent the formation of intracellular ice. In this project, a fundamentally new approach will be taken, involving the development of a generalized probability model of the incidence of intracellular ice formation (IIF) during cell freezing. Detailed cryomicroscopic experiments will be performed on a mammalian germplasm system to (1) establish the existence and magnitude of the cell supercooling tolerance, (2) develop the mathematical forms of a probability model for instantaneous and time-dependent IIF, and (3) determine the parametric effect of cryoprotectants on the supercooling tolerance and probability of IIF. Concepts from reliability theory will be used to develop a mathematical model of the probability of IIF and to synthesize various sources of randomness in cell freezing behavior. A reduced set of experiments will be performed on a plant cell system to test the universality of the modeling approach. The model will be used to explore the feasibility of innovative cooling protocols that could significantly increase cell survival rates. This project will result in a fundamental step forward in the development of engineering models of the response of cells to freezing, and in the creation of new analytical tools with which to design cryopreservation protocols. The development of the model will have a wide range of cryopreservation applications: human and animal sperm, oocytes, and embryos; organs and specialized tissues; plants shoot tips for asexually propagated species; and the conservation of the germplasm of endangered plant and animal species.