New Cell Desiccation Strategy Using Non-Isothermal Drying and Biophysical Models

描述：有效的活细胞保存技术在生物医学应用中是必要的，从体外受精到组织工程和细胞治疗，因为（非稳定）细胞的保质期短，对生物银行、长途运输甚至安全/质量测试造成了严重的限制。目前的保存策略是不够的，特别是对于敏感的细胞类型，如卵母细胞。例如，低温保存需要暴露于高浓度的渗透化学品中，这些化学品用作低温保护剂（cpa），但具有固有的细胞毒性，并且需要昂贵的液氮（LN2）冷却容器进行储存和运输。此外，低温保存的缓慢冷却方法不能令人满意地恢复健康卵母细胞，而最近的卵母细胞玻璃化方法需要不安全的LN2直接接触，不可靠的样品制备方法，以及更高的CPA浓度。另一种保存策略，即脱水（通过干燥），有可能将细胞稳定在更方便的储存温度下（例如，室温），但目前的干燥方法是有害的，因为在干燥过程中形成的扩散障碍使细胞暴露于长时间的渗透胁迫下；哺乳动物卵母细胞的干燥目前是不可能的。人类卵母细胞干燥过程的发展将允许保留癌症患者的生育能力，并为其他疾病提供不孕治疗选择，包括卵巢早衰、卵巢过度刺激综合征和多囊卵巢。该研究计划的长期目标是通过开发一种新颖的、数学上优化的干燥方法来满足对有效、低成本细胞保存技术的关键需求。这个开发（R21）应用程序的目标是证明的可行性

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.