This proposal is an extension of previous work to more clearly define intracellular and intercellular ice formation when cells are physically in contact. New experimental techniques including high speed video microscopy to assess IIF at unprecedented speed (30,000 frames/second), as well as a new (untried) method for measuring water transport using fluorescence quenching are proposed. Completion of the proposed work would advance the knowledge and understanding of IIF initiation, growth, and propagation and the effects of thermodynamic and non-thermodynamic variables on these processes. While the experimental approach uses two-cell and monolayer cultures, theoretical modeling is expected to elucidate optimal approaches for the cryopreservation of tissues and tissue engineered constructs. The work includes an implementation of an open-source project for cryobiology software developed in the course of the research including software previously developed by our group, as well as software developed by other research groups. Knowledge gained from the completion of this work would benefit the field of cryobiology, tissue engineering, and cryosurgery.
