Novel Disaccharide-Based Cryopreservation Approach for Stable, -80°C Storage of Stem Cells

摘要 细胞的长期冷冻保存需要使用冷冻保护剂（CPA）。二甲亚砜（DMSO） 是目前保存细胞的“金标准”CPA，已被用于长期保存细胞达半个世纪。而 DMSO被认为对干细胞无毒，但它确实会改变细胞生理学，特别是当储存溶液变热时。 更严重的是在移植患者中报告的归因于DMSO毒性的副作用。其他问题 DMSO是超低温的要求，这大大增加了基础设施的需求， 储存和运输费用。我们正在开发基于海藻糖的新型注射CPA替代DMSO 用于储存干细胞以及其他细胞类型，在非低温温度（例如≤-80 ° C）下也是稳定的。 海藻糖是一种非还原性双糖，具有特殊的稳定和保存细胞的能力 和细胞结构。然而，与可自由膜扩散的DMSO不同，海藻糖 哺乳动物细胞不能自然吸收。我们已经克服了这一限制，使用ATP激活的嘌呤能 受体P2 X7。本文提出的工作将改进使用海藻糖的冷冻保存方法， 最佳冷冻和储存参数。如果成功，这种方法将大大降低成本， 冷冻保存许多不同细胞类型和组织所固有的技术障碍。

ABSTRACT Long-term cryopreservation of cells requires the use of cryoprotectant agents (CPAs). Dimethysulfoxide (DMSO) is currently the “gold standard” CPA, which has been used for half a century to preserve cells long-term. While DMSO is considered non-toxic to stem cells, it does alter cell physiology, especially as storage solutions warm. More serious is the reported side effects in transplant patients attributed to DMSO toxicity. Additional issues with DMSO are requirements for ultralow temperatures which significantly increases the infrastructure needs and costs for storage and shipping. We are developing novel injectable CPA alternative to DMSO based on trehalose for banking stem cells as well as other cell types, that is also stable at non-cryogenic temperatures (e.g. ≤-80°C). Trehalose is a non-reducing disaccharide that possesses an exceptional ability to stabilize and preserve cells and cellular structures during freezing. However, unlike DMSO, which is freely membrane diffusible, trehalose is not naturally taken up by mammalian cells. We have overcome this limitation using the ATP-activated purinergic receptor P2X7. The work proposed here will refine cryopreservation methods using trehalose and establish optimal freezing and storage parameters. If successful, this method will significantly reduce the costs and technical hurdles inherent to cryopreservation of many different cell types and tissues.