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

细胞为基础的治疗出现了一个关键方面的现代医疗保健再生医学和输血。不幸的是，目前的冷冻保存方案仍然是次优的，限制了这种治疗的有效性。冷冻保存过程中细胞活力的丧失已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注。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在冻结和解冻过程中冰再结晶对细胞的损伤。由于缺乏抑制这一过程的实际策略，对这一过程的详细了解对自然科学至关重要。这个多学科的提案将使用体外方法来解决这个问题，这些方法依赖于先进的光谱（共聚焦拉曼）和现代细胞/生物物理技术（纳米颗粒跟踪，ektacymetry分析，低温显微镜等）。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、自组装和水化性能）的相关性；2)研究这些分子对细胞膜、微粒（MP）形成、冰的成核和细胞内冰的重结晶的体外细胞效应。从了解这些抑制剂如何发挥作用中获得的知识将大大促进我们对冰再结晶的理解（包括在细胞存在和不存在的情况下）。这些知识将与其他过程直接相关，如生物矿化和钙化，并导致新方法的产生来控制这些生理上重要的过程。

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.