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.
