Trehalose Cryopreservation of Human Mesenchymal Stem Cells from Cord Tissue

Adequate hypothermic storage of human mesenchymal stem cells (hMSCs) is of fundamental importance since they have been explored in several regenerative medicine initiatives. However, the actual clinical application of hMSCs necessitates hypothermic storage for long periods, a process that requires the use of non-toxic and efficient cryo-reagents capable of maintaining high viability and differentiating properties after thawing. Current cryopreservation methods are based on cryoprotectant agents (CPAs) containing dimethylsulphoxide (DMSO), which have been shown to be toxic for clinical applications. In this study, we describe a simple and effective trehalose (TRE)-based solution to cryo-store human umbilical cord-derived MSCs (UC-MSCs) in liquid nitrogen. Cells viability, identity, chromosomal stability, proliferative and migration capacity, and stress response were assessed after cryopreservation in TRE as CPA, testing different concentrations by itself or in combination with ethylene glycol (EG). Here we show that TRE-stored UC-MSCs provided lower cell recovery rates compared with DMSO-based solution, but maintained good functional properties, stability, and differentiating potential. The best cell recovery was obtained using 0.5 M TRE with 10% EG showing no differences in the osteogenic, adipogenic, and chondrogenic differentiation capacity. A second cycle of cryopreservation in this TRE-based solution had no additional impact on the viability and morphology, although slightly affected cell migration. Furthermore, the expression of the stress-related genes, <i>HSPA1A</i>, <i>SOD2</i>, <i>TP53</i>, <i>BCL-2</i>, and <i>BAX</i>, did not show a higher response in UC-MSCs cryopreserved in 0.5 M TRE + 10% EG compared with DMSO. Together these results, in addition to ascertained therapeutic properties of TRE, provide sufficient evidence to consider TRE-based medium as a low-cost and efficient solution for the storage of human UC-MSCs cells and potentially substitute DMSO-based cryo-reagents.