As hepatocyte-based therapies to treat liver failure are near to bear tangible fruit in the form of clinical applications, the fundamental issues associated with the long-term preservation of hepatocytes take a front line position for successful translation of these new technologies from bench-to-bed side. 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 <-80fC) 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. The dry state storage is based on removing water to achieve a glassy (i.e. amorphous or vitrified) state in and around cells at ambient temperatures. The glassy state is known to have an exceedingly high viscosity (>1012 Pa) that may inhibit the chemical, biological, and physical processes that lead to cell deterioration. Dried storage overcomes many of the problems associated with cryogenic storage including the transport issues, thermal fluctuations during cryogenic storage, sample size and weight, regulatory problems, cryoprotectant removal, among others. The evidence suggests that the survival of mammalian cells in the dry state is a complex phenomenon that requires control of the cell microenvironment and metabolism through the use of physicochemical and biological approaches in order to mimic the essential aspects of desiccation tolerance observed in nature. To this end, we formulated four distinct, but interrelated, Specific Aims.
In Specific Aim 1, we will control the microenvironment of hepatocytes during desiccation and long-term storage in order to minimize non- uniformities leading to cell death and damage.
In Specific Aim 2, we will study metabolic preconditioning of hepatocytes in order to improve survivorship in dry state.
In Specific Aim 3, we will characterize the biological stress response program during preconditioning and recovery from desiccation using high-throughput living cell arrays.
In Specific Aim 4, we will test the efficacy of desiccated cells in small animal models of liver failure. With the advancements being made in tissue engineering, regenerative medicine, and stem cell biology, the clinical demand for effective long term storage methods for cells and tissues will continue to increase for many different tissue types, including hepatocytes and liver. Desiccation of mammalian cells is a very attractive alternative strategy inspired from anhydrobiotic organisms that has the potential to truly disseminate these powerful technologies to medical centers, hospitals, and physicians'offices. For the treatment of acute and chronic liver failure, there is a critical need for both temporary and permanent modes of liver support. Hepatocyte-based liver support systems offer a viable alternative for both of these modalities but such a program relies heavily on a consistent, abundant, and readily available supply of hepatocytes. Thus proper hepatocyte storage techniques are of utmost importance if cell-based therapies are ever to become feasible.
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