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)。二甲基亚砜(DMSO) 是目前的“黄金标准”注册会计师，它已经使用了半个世纪，以长期保存细胞。而当 DMSO被认为对干细胞无毒，它确实会改变细胞的生理，特别是在储存溶液变暖的情况下。 更严重的是，据报道，移植患者的副作用可归因于DMSO毒性。有关的其他问题 DMSO是对超低温的要求，这显著增加了基础设施需求，并 储存和运输成本。我们正在开发基于海藻糖的新型可注射CPA替代DMSO 对于银行干细胞以及其他类型的细胞，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二糖，具有稳定和保存细胞的特殊能力 以及冷冻过程中的细胞结构。然而，与DMSO不同的是，DMSO是自由的膜扩散，海藻糖是 不被哺乳动物细胞自然吸收的。我们已经使用ATP激活的嘌呤能克服了这一限制 受体P2X7。这里提出的工作将改进使用海藻糖的冷冻保存方法，并建立 最佳的冷冻和储存参数。如果成功，这种方法将大大降低成本和 冷冻保存许多不同类型的细胞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.