Understanding Ice Recrystallization Inhibitors - Design, Synthesis and Mechanism(s) of Action

在再生医学和输血中，基于细胞的疗法成为现代医疗保健的一个关键方面。不幸的是，目前的冷冻保存方案仍然不是最优的，并且限制了这种疗法的有效性。在低温保存过程中细胞活力的丧失被证明会显著降低祖细胞的效力，如用于造血干细胞(HSC)移植和红细胞(RBC)输注的祖细胞，导致植入不良或需要额外输血。最近的研究表明，解冻后细胞活力下降和功能丧失的最重要原因是冷冻和解冻过程中冰重结晶对细胞的损害。对这一过程的详细了解是自然科学的基础，因为缺乏抑制这一过程的实际战略。这项多学科的提案将使用依赖于先进的光谱学(共聚焦拉曼)和现代细胞/生物物理学技术(纳米颗粒跟踪、电子显微镜分析、冷冻显微镜等)的体外方法来解决这一问题。具体目标是：1)研究我们的新型冰重结晶抑制剂(IRIS)的能力与其物理参数(疏水性、自组装和水化特性)的关系，以及2)研究这些分子在体外对细胞膜、微粒(MP)的形成、冰的成核和细胞内冰的重结晶的细胞效应。通过了解这些抑制物如何发挥作用所获得的知识将极大地促进我们对冰重结晶的理解(无论是在细胞存在还是不存在的情况下)。这一知识将与生物矿化和钙化等其他过程直接相关，并导致产生控制这些重要生理过程的新方法。 重要性/影响： 我们依靠先进的光谱学(共聚焦拉曼)和现代细胞/生物物理技术(纳米颗粒跟踪、电子细胞仪分析、冷冻显微镜等)使用体外策略的综合方法。研究小分子虹膜将加速了解这些抑制剂是如何发挥作用的。此外，新的和改进的冷冻保护剂的设计是及时的，将解决与再生医学、细胞治疗和组织工程领域的技术相关的许多储存问题。

Cell-based therapeutics emerged as a critical aspect of modern healthcare in regenerative medicine and transfusion. Unfortunately, current cryopreservation protocols remain suboptimal and limit the effectiveness of such therapies. Loss of cell viability during cryopreservation has been shown to dramatically reduce potency of progenitor cells such as those used in hematopoietic stem cell (HSC) transplantation and red blood cell (RBC) transfusion resulting in poor engraftment or a need for additional transfusions. Recent studies have indicated that the most significant cause of decreased post-thaw cell viability and loss of function is cellular damage from ice recrystallization during freezing and thawing. A detailed understanding of this process is fundamental to the natural sciences since practical strategies to inhibit it are lacking. This multidisciplinary proposal will address this issue using in vitro approaches relying on advanced spectroscopic (confocal Raman) and modern cellular/biophyscial techniques (nanoparticle tracking, Ektacytometric analysis, cryomicroscopy etc.). Specific objectives are; 1) study and correlate the ability of our novel ice recrystallization inhibitors (IRIs) to their physical parameters (hydrophobicity, self-assembly and hydration properties) and 2) study the in vitro cellular effects of these molecules on cellular membranes, microparticle (MP) formation, nucleation of ice and the recrystallization of intracellular ice. The knowledge gained from understanding how these inhibitors function will significantly advance our understanding of ice recrystallization (both in the presence and absence of cells). This knowledge will be directly relevant to other processes such as biomineralization, and calcification and lead to the generation of novel methods to control these physiologically important processes. Significance/Impact: Our integrated approach of using in vitro strategies relying on advanced spectroscopic (confocal Raman) and modern cellular/biophyscial techniques (nanoparticle tracking, Ektacytometric analysis, cryomicroscopy etc.) to study small molecule IRIs will expedite an understanding of how these inhibitors function. In addition, the design of new and improved cryoprotectants is timely and will address many of the storage problems associated with technologies in the areas of regenerative medicine, cellular therapy and tissue engineering.