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

产品说明：活细胞的有效保存技术在从体外受精到组织工程和细胞治疗的生物医学应用中是必要的，因为（非稳定的）细胞的短保质期对生物库、长途运输甚至安全/质量测试施加了严重的限制。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，冷冻保存需要暴露于高浓度的渗透性化学品，其用作冷冻保护剂（CPA）但具有固有的细胞毒性，并且需要由液氮（LN 2）冷却的昂贵的容器用于储存和运输。此外，缓慢冷却的方法冷冻保存产生不满意的恢复健康的卵母细胞，而最近的卵母细胞玻璃化方法需要不安全的直接接触液氮2，不可靠的样品制备方法，和更高的CPA浓度。另一种保存策略，脱水（通过干燥），有可能在更方便的储存温度下稳定细胞（例如，室温），但目前的干燥方法是有害的，因为细胞由于干燥过程中形成的扩散屏障而暴露于长时间的渗透应力;哺乳动物卵母细胞的干燥目前是不可能的。人类卵母细胞干燥程序的开发将允许保留癌症患者的生育能力，并为其他疾病状态提供不孕症治疗选择，包括卵巢早衰，卵巢过度刺激综合征和多囊卵巢。拟议研究计划的长期目标是通过开发一种新颖的数学优化干燥方法来满足有效，低成本细胞保存技术的关键需求。本开发（R21）应用的目的是证明以下方法的可行性： 在小鼠和人类卵母细胞中的拟议方法。这一目标将使用模拟引导的方法，并通过利用细胞内和细胞外糖的独特保护特性来实现。研究人员以前曾使用这种策略来改善冷冻保存的结果。初步研究表明，卵母细胞可以干燥到5%的残留水分含量，而不会显著丧失功能， 假设卵母细胞可以安全地在-20 ° C（用于储存在常规冰箱中）或4 ° C（冰箱储存）的温度下通过使用非等温干燥方法在存在细胞内和细胞外糖、糖聚合物和少量（d0.5 M）渗透性CPA的情况下进入玻璃态。提出的非等温干燥方法是创新的，因为它规避了等温干燥固有的扩散障碍问题，从而允许更快，更少的破坏性脱水;其他创新包括开发仅使用最少量CPA的新型干燥解决方案，以及使用基于生物制药学的数学模型和计算机模拟来确定非等温干燥的最佳方案。这项研究意义重大，因为它的成功将（i）改变当前细胞保存的范式，（ii）为细胞治疗和组织工程中的细胞生物保存需求铺平道路。

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.