School of Science, College of STEM, RMIT University, Melbourne, Australia.
ARC Centre of Excellence in Exciton Science, School of Science, College of STEM, RMIT University, Melbourne, Australia.
J Mater Chem B. 2022 Jun 22;10(24):4546-4560. doi: 10.1039/d2tb00573e.
Cryopreservation has facilitated numerous breakthroughs including assisted reproductive technology, stem cell therapies, and species preservation. Successful cryopreservation requires the addition of cryoprotective agents to protect against freezing damage and dehydration. For decades, cryopreservation has largely relied on the same two primary agents: dimethylsulfoxide and glycerol. However, both of these are toxic which limits their use for cells destined for clinical applications. Furthermore, these two agents are ineffective for hundreds of cell types, and organ and tissue preservation has not been achieved. The research presented here shows that deep eutectic solvents can be used as cryoprotectants. Six deep eutectic solvents were explored for their cryoprotective capacity towards mammalian cells. The solvents were tested for their thermal properties, including glass transitions, toxicity, and permeability into mammalian cells. A deep eutectic solvent made from proline and glycerol was an effective cryoprotective agent for all four cell types tested, even with extended incubation prior to freezing. This deep eutectic solvent was more effective and less toxic than its individual components, highlighting the importance of multi-component systems. Cells were characterised post-thawing using atomic force microscopy and confocal microscopy. Molecular dynamics simulations support the biophysical parameters obtained by experimentation. This is one of the first times that this class of solvents has been systematically tested for cryopreservation of mammalian cells and as such this research opens the way for the development of potentially thousands of new cryoprotective agents that can be tailored to specific cell types. The demonstrated capacity of cells to be incubated with the deep eutectic solvent at 37 °C for hours prior to freezing without significant loss of viability is a major step toward the storage of organs and tissues.
冷冻保存技术促进了许多突破，包括辅助生殖技术、干细胞疗法和物种保护。成功的冷冻保存需要添加抗冻保护剂来防止冷冻损伤和脱水。几十年来，冷冻保存主要依赖于两种主要的试剂：二甲基亚砜和甘油。然而，这两种试剂都具有毒性，限制了它们在临床应用细胞中的使用。此外，这两种试剂对数百种细胞类型都无效，器官和组织的保存也没有实现。本研究表明，深共晶溶剂可用作抗冻保护剂。研究了六种深共晶溶剂对哺乳动物细胞的抗冻保护能力。测试了这些溶剂的热性能，包括玻璃化转变温度、毒性和对哺乳动物细胞的渗透性。脯氨酸和甘油形成的深共晶溶剂是四种测试细胞类型的有效抗冻保护剂，即使在冷冻前延长孵育时间也是如此。这种深共晶溶剂比其单独的成分更有效、毒性更小，突出了多组分系统的重要性。通过原子力显微镜和共聚焦显微镜对解冻后的细胞进行了表征。分子动力学模拟支持通过实验获得的生物物理参数。这是首次系统地测试这类溶剂对哺乳动物细胞的冷冻保存，因此这项研究为开发可能数千种可针对特定细胞类型定制的新型抗冻保护剂开辟了道路。细胞在 37°C 下与深共晶溶剂孵育数小时后再进行冷冻而不会显著丧失活力的能力，这是器官和组织储存的重要一步。
