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.
The development of cryopreservation technology for organs and tissues would prevent the degradation of such living materials during transportation, safety testing, and storage prior to transplantation. Unfortunately, the freezing of biological tissue can result in significant damage caused by the crystallization of water that is trapped in the tissue’s cells. Recent discoveries under the present project have led to an improved understanding of the events that trigger intracellular ice formation during tissue cryopreservation. The knowledge gained from these investigations is making possible the development of computer simulations that can predict the extent of tissue cryoinjury during the freezing of organs, grafts, and tissue engineered medical products. Such simulations can then be used to guide the design of novel cryopreservation methods that minimize cell damage. The ultimate objective of this research program is to increase the quality and quantity of organs and engineered tissues available for potentially life-saving transplantation procedures. To determine the causes for cell freezing in tissues, state-of-the-art experimental techniques were adapted for use in investigations of tissue cryopreservation. For example, using miniaturized rubber stencils to pattern a glass surface with chemicals that alternately promote or inhibit cell attachment, engineers were able to create microscopic tissue constructs with exact control of cell size, shape, and the degree of interaction between neighboring cells. This novel approach allowed the researchers to study the role of cell geometry and cell-cell interactions on the tissue’s response to freezing. In addition, the research team used a unique high-speed video cryomicroscopy system to capture video of intracellular ice crystal formation events that are literally a thousand times faster than the blink of an eye. Analyzing the resulting images, the project staff identified several external factors that can trigger cell freezing. Specific findings resulting from the funded research include evidence for, and mathematical models of, several hitherto-unknown mechanisms whereby intracellular ice formation is triggered in tissue engineered constructs. For example, high-speed video evidence suggests that external ice crystals extend protrusions that can penetrate into water pockets at the interface between neighboring cells, or between an adherent cell and the surface to which it is attached. The appearance of these penetrating ice structures is associated with subsequent ice crystallization of the supercooled cell interior. A second mechanism of intracellular ice formation, observed in cells that have spread out on flat surfaces, causes intracellular ice formation to be initiated at sites along the perimeter of the cell’s outermost edge. Some experimental evidence suggests that this phenomenon is related to the structure of protein complexes that form along the cell perimeter as the cell attaches itself to a surface. Other outcomes of the project include the development and characterization of a fluorescence-based technique to measure the rate at which water can move through the membrane of cells that are growing on a surface (whereas conventional measurement methods require the detachment of cells from their growth scaffold, making it impossible to analyze intact tissue). It was found that in cells from blood vessels and from pancreatic islets, water moved more readily out of cells when grown as tissue constructs on surfaces than after detachment of cells. Computer simulations predicted that less intracellular water would be trapped during freezing of intact cell monolayers than during freezing of suspensions of isolated cells, which indicates that damage due to crystallization of cell water will be less likely in the former case. The insights and data gained from this research is anticipated to lead to improved cryopreservation technology for tissue engineered medical products. Such advances are necessary to make possible the economical mass production of tissue and organ equivalents, which has the potential to solve organ shortage crisis currently affecting over 100,000 Americans who are on transplant waiting lists.
