Multicomponent solutions for the preservation of cell therapy products

项目摘要 一种新颖的、受生物学启发的策略将用于改善心肌细胞的无 DMSO 保存 源自以聚集体形式培养的人类诱导多能干 (hiPS) 细胞。多细胞系统 对传统的保存方法反应不佳。在上一个融资周期中，出现了差异化演变 使用糖、糖醇和糖的组合来优化细胞的保存算法 氨基酸。这种方法模仿了植物和其他简单生物的生存策略 环境压力。表征多组分渗透剂溶液行为的研究 证明这些溶液中存在的渗透剂相互作用以改变水的行为 在冷冻过程中，这表现在分子水平（氢键）以及 微观结构。渗透剂还与细胞中的关键生物结构相互作用以稳定它们。低 温度拉曼光谱研究表明，多细胞系统对 过冷度（结冰温度与结冰温度之间的温差） 在细胞外溶液中形成）。当我们从单细胞过渡到聚集体时，我们再次寻找 向大自然寻求灵感。林蛙使用渗透调节剂、抑制细胞代谢和 最大限度地减少过冷，因为它适应冬季的冰冻条件。我们正在提出类似的方法 改善心肌细胞聚集体的保存。申请中描述的研究特征 iPSC 来源的心肌细胞在定型心脏祖细胞阶段以及当 形成完全分化的多细胞心脏结构。过冷度对解冻后的影响 还将确定完全分化的多细胞心脏结构的恢复。的影响 抑制细胞代谢对多细胞构建体解冻后恢复的影响也将被量化。上一个 从根本上讲，这些研究将增进我们对更复杂的冻结行为的理解 多细胞系统。在应用层面上，拟议的研究有可能保存细胞 通过使用自然启发的策略与 hiPS 细胞分化。

Project Abstract A novel, biologically inspired strategy will be used to improve DMSO-free preservation of cardiomyocytes derived from human induced pluripotent stem (hiPS) cells cultured as aggregates. Multicellular systems respond poorly to conventional preservation methods. In the previous funding cycle, a differential evolution algorithm was used to optimize the preservation of cells using combinations of sugars, sugar alcohols and amino acids. This approach mimicked the strategies of plants and other simple organisms that survive environmental stresses. Studies characterizing the behavior of multicomponent osmolyte solutions demonstrated that osmolytes present in these solutions interact with each other to modify the behavior of water during freezing which manifests itself at a molecular level (hydrogen bonding) as well as changes in microstructure. The osmolytes also interact with critical biological structures in the cell to stabilize them. Low temperature Raman spectroscopy studies demonstrated that multicellular systems are sensitive to undercooling (the temperature difference between the freezing temperature and the temperature at which ice forms in the extracellular solution). As we transition from single cells to aggregates, once again, we are looking to nature for inspiration. The wood frog uses combinations of osmolytes, suppressed cell metabolism, and minimizes undercooling as it adapts to freezing conditions in the winter. We are proposing a similar approach to improving preservation of cardiomyocyte aggregates. The studies described in the application characterize the freezing response of iPSC-derived cardiomyocytes at the committed cardiac progenitor stage and when formed into fully differentiated, multicellular cardiac constructs. The influence of undercooling on post thaw recovery of fully differentiated multicellular cardiac constructs will also be determined. The influence of suppressing cell metabolism on post thaw recovery of multicellular constructs will also be quantified. On a fundamental level, these studies will advance our understanding of the freezing behavior of more complex multicellular systems. On an applied level, the proposed investigation has the potential to preservation of cells differentiated from hiPS cells through the use of naturally inspired strategies.