With the advancements being made in tissue engineering, cell transplantation, stem cell biology, and gene therapy, the clinical demand for effective long-term storage methods for cells and tissues will continue to increase. We propose to develop novel methods to biostabilization of mammalian cells for long-term preservation in a desiccated state at ambient temperature. In nature, many animals and organisms down regulate their metabolism and may enter into a state of stasis by either desiccation through removal of water from their cells (i.e., anhydrobiosis) or by a developmentally programmed arrest under full hydration (i.e., diapause). The ability to enter diapause prior to desiccation is crucial for the survivorship of many organisms that undergo natural states of dormancy. Furthermore, a common theme is that desiccation-tolerant animals accumulate large amounts of disaccharides, especially trehalose and sucrose. These sugars provide protective effects by forming stable sugar glasses at high water contents, and by stabilizing biological membranes and proteins through direct interaction with polar residues. We, therefore, hypothesize that metabolic preconditioning of mammalian cells to induce diapause-like state followed by controlled drying, storage, and rehydration conditions (i.e., physicochemical, biochemical, and metabolic) can be used to achieve desiccation tolerance in mammalian cells and tissues. To this end, our 3 distinct, but interactive, specific aims are: 1) To develop optimal physicochemical conditions to stabilize desiccated cells. 2) To metabolically precondition mammalian cells to improve survivorship during storage. 3) To develop metabolic and biophysical strategies to accelerate recovery of desiccated cells. This project is one of the first attempts to apply engineering and quantitative concepts to achieving anhydrobiotic state in mammalian cells. It provides a systems view of the metabolic and cellular changes a cell encounters before, during, and after desiccation. This project is inspired by nature and it uses engineering concepts and approaches to translate nature's solution to long-term storage or """"""""suspended animation"""""""" for mammalian systems. The proposed studies will significantly impact on human health by providing a solution to the problem of providing long-term storage of blood cells, stem cells, tissue engineered products, and cell-based biosensors for use in regenerative medicine, tissue engineering, and bioterrorism. In the short term, it will help increase the treatment modalities available to liver failure by providing stable, long-term stabilized cells for bioartificial liver assist devices. The longer-range outcome of the proposed research is to translate the information gained from these studies into whole organ preservation.
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