Our overall goal is to develop long-term hepatocyte storage strategies that can be used for the treatment of liver failure. Although cell stasis is routinely achieved in nature by anhydrobiotic organisms through desiccation at ambient temperatures, the only existing strategy for long-term storage of mammalian cells is cryopreservation. Cryopreservation relies on cryogenic temperatures (typically < -80 degrees C) in order to halt all chemical reactions that result in cell death during storage. On the other hand, the pharmaceutical industry has made significant strides in storing proteinaceous drugs, liposomes, membranes, and viral particles in dry state using various small sugar molecules as stabilizers. Sugars, such as disaccharides, enter glassy phase at ambient temperatures at low moisture levels (approximately 5%) and minimize molecular mobility. Our hypothesis is that molecular mobility in the desiccated state needs to be completely prevented to stop deterioration of the cells and the cellular injury during dried storage. Stabilizers such as sugars, therefore, are needed to halt molecular mobility in order to achieve stability and long-term viability of cells in a dried state. We will test our hypothesis is four distinct but interactive Specific Aims.
In Specific Aim #1, we will determine molecular mobility in dried cells in the glassy and near glassy state.
In Specific Aim #2, we will investigate the mechanisms of cell death associated with cellular desiccation and to develop strategies to modulate these processes to improve survival.
In Specific Aim #3, we will determine the stability of desiccated living cells and the key parameters affecting shelf life.
In Specific Aim #4, we will test the efficacy of desiccated cells in small animal models of liver failure. The proposed studies will be carried out using several key sugars including trehalose, sucrose, raffinose, stachyose, and mixtures thereof. 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 for many different tissue types, including liver. Desiccation of mammalian cells is a very attractive alternative strategy that has the potential to truly disseminate these powerful technologies to medical centers, hospitals, and physicians' offices.
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