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、冷却速率和防止细胞内冰形成的最终预淬火温度。在这个项目中，将采取一种全新的方法，涉及在细胞冻结期间细胞内冰形成（IIF）发生率的广义概率模型的发展。详细的低温显微实验将在哺乳动物种质系统上进行，以：(1)建立细胞过冷耐受性的存在和大小，(2)建立瞬时和时间相关的IIF概率模型的数学形式，以及(3)确定冷冻保护剂对过冷耐受性和IIF概率的参数效应。可靠性理论的概念将用于建立IIF概率的数学模型，并综合细胞冻结行为的各种随机性来源。一组简化的实验将在植物细胞系统上进行，以测试建模方法的普遍性。该模型将用于探索能够显著提高细胞存活率的创新冷却方案的可行性。该项目将在细胞对冷冻反应的工程模型的发展方面迈出根本性的一步，并在设计冷冻保存方案的新分析工具的创建方面取得进展。该模型的发展将具有广泛的冷冻保存应用：人类和动物精子、卵母细胞和胚胎；器官和专门组织；植物无性繁殖种的茎尖；濒危动植物种质资源的保护。

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.