DESCRIPTION: Effective preservation technology for living cells is necessary in biomedical applications ranging from in vitro fertilization to tissue engineering and cell therapies, because the short shelf-life of (non-stabilized) cells imposes severe limitations on biobanking, long-distance transportation, and even safety/quality testing. Current preservation strategies are inadequate, especially for sensitive cell types such as oocytes. For example, cryopreservation requires exposure to high concentrations of penetrating chemicals that are used as cryoprotectants (CPAs) but have inherent cytotoxicity, and requires expensive containment cooled by liquid nitrogen (LN2) for storage and transportation. Moreover, slow-cooling approaches to cryopreservation yield unsatisfactory recovery of healthy oocytes, whereas recent oocyte vitrification approaches require unsafe direct contact with LN2, unreliable sample preparation methods, and much higher CPA concentrations. An alternative preservation strategy, anhydrobiosis (via desiccation), has the potential to stabilize cells at more convenient storage temperatures (e.g., room temperature), but current approaches to desiccation are damaging, because cells are exposed to prolonged osmotic stresses due to diffusion barriers that develop during drying; desiccation of mammalian oocytes is not currently possible. The development of desiccation procedures for human oocytes would allow preservation of fertility for cancer patients, and offer infertility treatment options for other disease states including premature ovarian failure, ovarian hyperstimulation syndrome, and polycystic ovary. The long-term goal of the proposed research program is to meet the critical need of effective, low-cost cell preservation technology by developing a novel, mathematically optimized approach to desiccation. The objective of this developmental (R21) application is to demonstrate feasibility of the proposed method in mouse and human oocytes. This goal will be achieved using a simulation- guided approach, and by taking advantage of unique protective properties of intra- and extracellular sugars. The investigators have previously used this strategy to improve cryopreservation outcome. Informed by preliminary studies indicating that oocytes can be dried to as little as 5% residual moisture content without significant loss of functionality, the central hypothesis is that oocytes can safely be brought into a glassy state at temperatures of -20?C (for storage in a conventional freezer) or 4?C (refrigerator storage) by using a non-isothermal desiccation approach in the presence of intra- and extracellular sugars, sugar polymers, and small amounts (d0.5 M) of a penetrating CPA. The proposed non-isothermal desiccation approach is innovative, because it circumvents the diffusion barrier problem that is inherent to isothermal desiccation, thus allowing faster, less damaging dehydration; other innovations include the development of novel desiccation solutions that use only minimal amounts of CPA, and the use of biophysics-based mathematical models and computer simulations to identify the optimal protocols for non-isothermal desiccation. The proposed research is significant, because its success will (i) shift current paradigms of cell preservation, and (ii) pave the way for meetin biopreservation needs in cell therapy and tissue engineering.
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