Multicomponent solutions for the preservation of cell therapy products

项目摘要 一种新颖的、受生物启发的策略将用于改善心肌细胞的无DMSO保存 来源于作为聚集体培养的人诱导多能干（hiPS）细胞。多细胞系统 对传统的保存方法反应不佳。在上一个供资周期， 使用算法来优化使用糖、糖醇和葡萄糖的组合的细胞保存。 个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 证明了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，它表现在分子水平上（氢键）， 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（结冰温度和冰的温度之间的温差） 在细胞外溶液中形成）。当我们从单细胞过渡到聚合体时，我们再次看到， 到大自然中寻找灵感树蛙使用渗透调节剂，抑制细胞代谢， 最大限度地减少过冷，因为它适应冬季的冰冻条件。我们提出了一个类似的方法 改善心肌细胞聚集体的保存。申请中描述的研究表征了 iPSC衍生的心肌细胞在定向心脏祖细胞阶段的冷冻反应，以及 形成完全分化的多细胞心脏结构。过冷度对解冻后的影响 还将测定完全分化的多细胞心脏构建体的恢复。的影响 还将定量对多细胞构建体解冻后恢复的细胞代谢的抑制。上 这些研究将推进我们对更复杂的冻结行为的理解， 多细胞系统在应用水平上，所提出的研究具有保存细胞的潜力 通过使用自然启发的策略从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.