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

摘要

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.