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、冷却速率以及防止细胞内冰形成的最终预淬火温度。在该项目中，将采取一种全新的方法，涉及开发细胞冷冻过程中细胞内冰形成（IIF）发生率的广义概率模型。将对哺乳动物种质系统进行详细的低温显微镜实验，以（1）确定细胞过冷耐受性的存在和大小，（2）开发瞬时和时间依赖性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。从可靠性理论的概念将被用来开发一个数学模型的概率IIF和综合各种来源的随机性细胞冷冻行为。一组减少的实验将在植物细胞系统上进行，以测试建模方法的通用性。该模型将用于探索创新冷却方案的可行性，这些方案可以显着提高细胞存活率。该项目将导致在开发细胞对冷冻的反应的工程模型方面迈出基本的一步，并创造新的分析工具来设计冷冻保存方案。该模型的开发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;无性繁殖物种的植物茎尖;以及濒危植物和动物物种种质的保护。

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.