基于可降解纳米海藻糖的人诱导多功能干细胞冷冻保存研究

Human induced pluripotent stem cells (HiPS cells) has broad prospects in the application of cell medicine, however, cryopreservation of this sensitive cells is still facing big challenge, such as low survival rate and ice injury on cell stemness. The conventional cryoprotectant dimethyl sulfoxide is highly toxic at body temperature and has been found to induce differentiation in a large number of stem cells. Trehalose is a natural non-toxic cryoprotectant, showing a good ability to stabilize the cell membrane and protein during freezing. It can inhibit lethal intracellular ice growth and reduce cell damage, in addition, it can effectively prevent the differentiation of stem cells. However, trehalose itself is difficult to penetrate the cell membrane, therefore, can not be used to maximize the effectiveness of cryopreservation. On the basis of the previous work, we proposed a biodegradable nanotrehalose system for HiPS cells, which can achieve an effective intracellular delivery of trehalose. A series of in-depth research and experiments will be conducted in this project, including investigation on the basic thermal properties of biodegradable nanotrehalose, ice crystal growth mechanism during phase change, trehalose diffusion and transfer, screening the optimal nanotrehalose platform for HiPS cryopreservation. It is worth noting that the development of this project has important practical significance to achieve a green cell cryopreservation technology.

人诱导多功能干细胞（HiPS细胞）在细胞医学应用领域具有十分广阔的应用前景，但是这种敏感性细胞却面临着冷冻存活率低、复温后干细胞功能受损等巨大挑战。常规冷冻保护剂二甲基亚砜不仅毒性高，同时在复温后存在干细胞大量分化的现象。海藻糖作为天然无毒冷冻保护剂，具有在冷冻过程中稳定细胞膜和蛋白质的良好能力，可抑制致命的冰晶生长从而降低细胞损伤，并可有效防止干细胞分化。然而，海藻糖本身很难透过细胞膜，无法在低温下有效发挥保护作用。在前期工作基础上，申请人提出了用于HiPS细胞的可降解纳米海藻糖体系，有望实现细胞内无创高效地输送海藻糖。本项目将通过深入的实验研究，探明纳米海藻糖的基本热物性，揭示其在低温下抑制冰晶生长的作用机制，分析纳米海藻糖在细胞内外生物传热传质现象，筛选最佳的HiPS细胞纳米海藻糖冷冻保护方案。本项目的开展对实现精确化、绿色化细胞冷冻保存技术具有至关重要的现实意义。

人诱导多能干细胞(hiPSC)因其与胚胎干细胞相似的无限复制和分化潜能，一直是生物医学的研究热点。然而，hiPSC的应用却受制于保存技术，当前的hiPSC面临着冷冻存活率低，复温后干细胞功能受损等挑战，因此开发绿色、无毒、高效的冷冻保护剂非常重要。海藻糖广泛应用于替代有毒的渗透性保护剂，但是由于其难以渗透细胞膜发挥最佳的冷冻效果，近年来，很多研究人员尝试纳米海藻糖用于干细胞的慢速冷冻并获得了成功。在前期工作的基础上，项目负责人提出了用于hiPSC的纳米海藻糖体系，系统深入地研究了其保护作用。在项目执行过程中，我们通过对比实验首次探明了纳米海藻糖的热学特性和冰晶生长机理，结果表明纳米海藻糖具有降低冻结温度、减缓冰晶生长和抑制重结晶的作用。借助荧光辅助的方法定量地研究了hiPSC对于纳米海藻糖的摄取行为。通过低温显微法研究了hiPSC在纳米海藻糖影响下的渗透作用和胞内冰晶生长行为，结合理论模型获得了hiPSC的低温生物物理特性。最后，首次将纳米海藻糖用于hiPSC的慢速冷冻。结果表明，纳米海藻糖的引入为hiPSC的高效保存提供了一种安全可靠的方法，实验过程中获得的数据和结论有利于hiPSC的低温保存技术的进步，对发展精确化、绿色化细胞冷冻保存技术具有重要意义。